For AIR 2 kinase assays, GST AIR 2 was blended with GST CDC 48. 3 or GSTCDC48. 1 in kinase buffer supplemented with myelin basic protein for 15 min at room temperature. Responses were separated axitinib molecular weight by SDS PAGE, transferred to nitrocellulose, and g ATP development was determined by phosphoimaging. Protein packing was visualized by Ponceau S staining or by searching with GST, AIR 1, or AIR 2 specific antibodies. KodakID 3. 1 quantification pc software was employed to measure protein loading and increase. Phosphorylation of MYBP by AIR 2 or AIR 1 kinases in the presence or lack of CDC 48. 1 or CDC 48. 3 was calculated as, although AIR2 or AIR 1 autophosphorylation in the presence or lack of CDC 48. 1 or CDC 48. 3 was determined as. Embryos were collected from C. elegans hermaphrodites, and LAP/GFP CDC 48. 3) treated with get a handle on, air 2, or cdc 48. 3 and reared at 22_C as described previously. Embryos were washed and resuspended in lysis buffer and sonicated over ice. Following centrifugation, responded lysates were stored at _80_C and frozen in liquid nitrogen. Protein concentration Plastid was determined by Bradford assay. For immunoprecipitations, 400 mg embryo extract was incubated with 5 ml appreciation purified AIR 2 antibody for 3 hr at 4_C. Twenty microliters protein G Sepharose beads were added and the extract incubated at 4_C for yet another time. The beads were pelleted by low speed centrifugation and washed 3 times in lysis buffer minus NP 40. Trials were separated by SDS PAGE, transferred to nitrocellulose, and the membranes probed with AIR 2 and GFP specific antibodies. Western analysis was done as previously described. For the in vitro binding assays, 400 mM GST AIR 2 was treated with Prescission Protease to remove the GST tag. The cleaved Flupirtine AIR 2 protein was then mixed with GST CDC 48. 3 or GST CDC 48. 1 bound to glutathione beads and rocked over ice for 3 hr. Beads were washed by rocking in PBS+20 mM HEPES, 0. 2000 Triton X 100 at 4_C for 5 min and pelleted. Products were separated by SDS PAGE, utilized in nitrocellulose, and the membranes probed with GST and AIR 2 specific antibodies. To perform in vitro ATPase assays, 0. 5 mM GST CDC 48. 3, GST CDC 48. 1, and numerous GST CDC 48. 3 mutant proteins were blended with ATP and 100 ml assay buffer + 20 mM MgCl2, and incubated at 37_C for 15 min. Absorbance at 630 nm was measured employing a spectrophotometer as described by the manufacturer. Activity in get a handle on reactions without ATP was deduced from experimental reactions. Enzyme activity was determined centered on a standard curve generated from adding increasing levels of inorganic phosphate to the assays. Relative ATPase activity was calculated from three independent experiments.
the Bax/Bak poor MEFs stayed fully resistant, as assessed by both long term clonogenicity or temporary viability. Killing of Noxa indicating cells needed either Bax or Bak, but the killing was more effective in the current presence of both. Sensitization to ABT 737 by Noxa is not restricted to the MEFs. The myelomonocytic order Celecoxib mobile line FDC P1 turned out to be extremely resistant to treatment with ABT 737, but release of Noxa, ineffectual on it’s own, increased sensitivity over 2,000 collapse. In contrast, as predicted from the related binding profiles of ABT 737 and Bad, introduction of Bad didn’t increase sensitivity, nor did the inert Noxa mutant 3E. The sensitized cells died by apoptosis, whilst the loss in plasma membrane integrity expected caspase activity, and cell death was connected with release of cytochrome c from mitochondria. ABT 737 also caused Bax/Bak dependent cytochrome c release in vitro, but only if Mcl 1 had been neutralized with Noxa. We conclude that ABT 737 is really a genuine BH3 mimetic, because it induces Bax/Bak mediated cell killing, but that its selective binding report limits its cytotoxicity in certain cell types. We attribute resistant cells to be sensitized otherwise by Gene expression the ability of Noxa to its capacity to counteract prosurvival proteins perhaps not qualified by ABT 737. Lack of the latter in many cell types factors to Mcl 1 as an crucial predictor of responsiveness to ABT 737, although Noxa objectives equally Mcl 1 and A1. Having implicated Mcl 1, we next tried whether refractory human carcinoma cell lines might be sensitized by downregulating Mcl 1, by retroviral introduction of both Noxa or a particular human Mcl 1 short hairpin RNA. Immunoblots Bazedoxifene 198480-56-7 showed that Mcl 1 levels were substantially downregulated in both HeLa cervical epithelial cells and MCF 7 breast epithelial cells. Notably, both means of reducing the Mcl1 degree potently sensitized these cells to ABT 737 in colony formation assays. In striking contrast, when Mcl 1 levels were unperturbed, long haul growth wasn’t bothered by ABT 737. Notably, reintroduction of mouse mcl 1, which is not qualified by the individual mcl1 particular RNAi hairpin used, restored community creation, excluding the contribution of nonspecific goals. We next considered whether the drug may kill by directly activating Bax/Bak, as proposed for many BH3 only proteins. Immediate initial appeared impossible because most cell types include equally Bax and Bak and none the less withstand high levels of the drug without any apparent ill effects. Moreover, we established that ABT 737 does not bind Bax and, when utilized on cells, only triggered Bax to endure the conformational change that marks its activation if Mcl 1 have been inactivated with Noxa or by mcl 1 RNAi.
The rate of change in body weight was calculated utilizing the following formula: BW frazee W/W0 3 100, where W and W0 would be the body weights on a specific experimental day and on the very first day of treatment, respectively. All animal studies in this study were conducted in accordance with methods permitted by the Institutional Animal Care and Use Committee Fingolimod supplier of Chugai Pharmaceutical Co., Ltd. Xenograft tumors were removed, fixed in formalin, and embedded in paraffin. Immunostaining for phosphorylated ALK was done using phospho ALK antibody. Immunohistochemistry was performed utilizing the DISCOVERY XT automated staining platform. Total RNA was hybridized to Human Genome U133 Plus 2, and reverse transcribed, labeled, and extracted utilising the RNeasy package. 0 arrays according to the manufacturers directions. The term value for every single probe was calculated using the GC RMA algorithm. For quantitative RT PCR, RNA was amplified in QuantiFast Lymphatic system Multiplex RT PCR using a Universal probe collection and the LightCycler System. Glyceraldehyde 3 phosphate dehydrogenase served being an internal control. To evaluate the in vitro kinase assay of ALK, we created a GST labeled, kinase domain of ALK or the mutants by using a Bac to Bac Baculovirus Expression System in Sf 9 insect cells according to the companies standards. Mutant constructs were generated utilizing the QuikChange Site Directed Mutagenesis Kit. The cells were lysed in lysis buffer and centrifuged. Glutathione Sepharose 4B was incubated for 1 hr with the soluble fraction of the lysate and washed in buffer A. The proteins were eluted with elution buffer. The protein expression and purification were confirmed by SDS PAGE. The EML4 ALK gene and the L1196M were placed into pcDNA3. 1/ hygro vector. EML4 ALK L1196M was created using the QuikChange Site Directed Mutagenesis Kit and confirmed by resequencing histone deacetylase HDAC inhibitor the entire construct. Ba/F3 EML4 ALK and the L1196M cell lines were produced by transfecting Ba/F3 cells with pcDNA3. 1/hygro EML4ALK and the L1196M mutant using the NucleoFector unit, stable transfectants were then isolated from the cultured medium without IL 3. Protein crystallography was done by proteros biostructures GmbH. The kinase domain of human ALK was expressed in SF9 cells with a GST fusion draw, which was removed by protease cleavage during purification, the kinase domain was then purified applying affinity, size exclusion, and ion exchange chromatography. The purified protein was targeted to 20?40mg/ml and located at_80_Cuntil use. Crystals were obtained at 4_C from sitting drops using a reservoir answer by vapor diffusion. The crystals were shock frozen in liquid nitrogen after the addition of 22% ethyleneglycol. Diffraction data were collected at 90 K at beamline X06SA in SLS using a PILATUS 6M detector.
Multiple lines of evidence suggest that TR compounds induce apoptosis in cancer cells mainly through repression of MCL1 expression, including: upon treatment with Gefitinib ic50 compounds, MCL1 protein levels decreased rapidly and preceded caspase service, ectopic expression of physiological levels of MCL1 rescued cancer cells from TR compounds, despite the expression of other genes however being repressed, the pattern of TR substance sensitivity across a section of cancer cell lines closely mirrored the pattern of sensitivity of these cell lines to MCL1 knockdown by RNAi, of over 40,000 genomic characteristics tested, the top function that expected sensitivity to TR compounds was the minimal expression of BCL xL, which gives redundant purpose with MCL1, ectopic expression of BCL xL rescued cancer cells from TR compounds, MCL1 repression by TR compounds resulted in the release of proapoptotic protein BAK from MCL1, and Bak deficiency guarded cells from TR compounds. These results declare that the process of cell death induced by TR compounds is better explained by MCL1 inhibition. This suggested that a few of the widely used chemotherapeutic drugs such as anthracyclines may possibly preferentially repress MCL1 to induce Mitochondrion apoptosis in tumefaction cells. Though the antitumor effect of anthracyclines has long been thought to be related to the drugs inhibition of DNA topoisomerase II and a relationship between reduced TOP2A expression and anthracycline answer in ER negative breast cancer patients has been described, our data declare that their exercise could be largely explained by inhibition of transcription, leading most substantially to the repression of temporary MCL1 transcripts. While it is achievable that numerous mechanisms of Flupirtine action explain the antitumor aftereffects of anthracyclines, at least in the experimental cancer models examined here, anthracycline gene phrase consequences most reflected transcriptional inhibition in place of DNA topoisomerase II inhibition. Furthermore, the similar pattern of sensitivity of cell lines to MCL1 knockdown in comparison to anthracycline treatment is also in keeping with an transcriptional inhibitory effect. The anthracycline MCL1 connection that is further strengthened by last, our observation BCL xL expression is predictive of resistance to MCL1 repression both in model systems and in patients with breast cancer. We observe that the concentration of doxorubicin utilized in our studies approximates that noticed in human cancer cells. Doxorubicin encourages topoisomerase II mediated DNA cleavage only at low concentrations, while at doses greater than _0. 4 mM, topoisomerase II mediated DNA cleavage is lost. These data thus declare that at clinically relevant levels, anthracyclines act as transcriptional repressors, as opposed to DNA damaging agents.
In this study we show the translocation of FADD from the cytosol to the cell membrane of Jurkat Cabozantinib ic50 cell treated with PDTI or SBTI, as well as the activation of caspase 8. At the DISC, procaspase 8 is prepared and activated. These events usually are linked to the death receptor pathway, even though it can’t be eliminated that FADD functions in a receptor independent way, as in the situation of cycloheximide induced cell death in Jurkat cell. It must be taken into consideration that both PDTI and SBTI have well recognized lectin like properties, besides their trypsin and chymotrypsin inhibitory activity; so that it is not possible to consider that the induction of cell apoptosis is due simply to its antiprotease activity. Furthermore, it could be speculated that these inhibitors interact with glycoconjugates associated to the cell membrane, hence initiating the cell death Organism process. Extremely, SBTI was more potent than PDTI in inducing apoptosis of Jurkat cells, in contrast to their impact on Nb2 cells, where PDTI proved to be active at reduced levels. Another striking huge difference in behavior is their capacity to cause cell death of human low activated lymphocytes while mouse lymphocytes were only prone to apoptosis after stimulation with concanavalin A. This difference might be due to species specificity. However, a few reports describe different responses between spleen and blood lymphocytes. Hussain et al. Defined that swine spleen cells were less sensitive and painful to mitogeninduced growth than pure blood lymphocytes. Yet another survey shows the result of 2 Capecitabine clinical trial acetyl 4 tetrahydroxybutyl imidazole in rat, this compound paid down significantly both lymphocytes T and T in blood, however, not spleen lymphocytes. Nygaard and L?vik compared the consequence of a immunosuppressive drug, cyclophosphamide, on rat blood and spleen lymphocytes showing larger effects in blood lymphocytes than in spleen cells. These studies underline the main advantage of performing immunotoxicological reports using blood lymphocytes. If the apoptosis inducing aftereffect of these inhibitors is restricted to lymphoid cells to judge, PDTI and SBTI were examined on cervical adenocarcinoma, HeLa, and human cell lines, hepatocellular carcinoma, HepG2, and only SBTI showed some cytotoxic effects on these adherent cells. These answers are consistent with the higher efficiency of SBTI with respect to PDTI to induce apoptosis of Jurkat cells. Further studies are warranted to higher comprehend the molecular events mixed up in apoptosis induced by these trypsin inhibitors. KRAS variations occur in _20% of all cancers, with particularly high frequency in pancreatic. colorectal. and lung cancers.
Fluoxetine restricted IFN induced SMase action and activations of Fluoxetine is not only a SSRI but additionally as an ASM chemical. Such as the link between sph24 and D609, IFN induced SMase activity was inhibited by fluoxetine. As previously demonstrated It also blocked COX 2 protein levels, phospho Akt, and STAT levels as well as lowering of ERK activation. BI-1356 solubility As similar outcomes of D609 seen in STAT phophorylation, fluoxetine restricted IFN significantly increased the quantities of phospho STAT1 at Ser727 and phospho STAT3 at Ser727. In our study, we have demonstrated that inhibition of SMase adjusts IFN activated 5 HT uptake via ERK and STAT activation. Moreover, an Akt dependent route and COX 2 induction participated in an inhibition of ASM on IFN induced ERK and STAT activation. These results show that NSM and ASM apply differential signal pathways to thereby improve 5 HT uptake. Little is also known that service of SMase fits with monoamine uptake, although dopamine uptake is induced by NSM through regulation of intracellular calcium. Ceramide is generally accepted as a modulator of monoamine transporter function. The enhanced 5HT uptake induced Skin infection by ceramide is regulated by dopamine transporter since it does occur in the absence of 5 HT transporters in striatal synaptosomes prepared from para chloroamphetamine treated subjects, and it does not occur in hippocampal synaptosomes with largely lacking dopamine transporters. Furthermore, this increased uptake is attenuated by pretreatment with selective dopamine reuptake inhibitor methylphenidate. But, the precise mechanism with this function natural compound library continues to be uncertain. Within our study, we found that both SMase types have the effect of IFN induced 5 HT uptake via an ERK/STAT dependent process. Furthermore, we did not found dramatically inhibitory effect of myriocin, an effective inhibitor of serine palmitoyltransferase for the initial step in sphingosine biosynthesis on IFN caused 5 HT usage, which might show that de novo ceramide synthesis is not necessary with this process. Acid SMase activity is inhibited by several antidepressant drugs such as fluoxetine functionally in peripheral blood mononuclear cells along with in brain tissue. Inside our research, the SMase inhibitors also lowered 5 HT uptake via an ERK/STAT dependent process in IFN treated T cells. In center, acid SMase activity based on PBMC fits with the severity of depression, and this finding also implies that the increased activity of acid SMase may have used implications for synaptic transmission and especially improved 5 HT uptake in central nervous system. Accordingly, an inhibition of p SMase may result in a increase of the 5 HT focus in the synaptic area.
Bax is the crucial amplifier of the extrinsic apoptosis, the special access level for the intrinsic apoptotic signaling, and the chemical which allows bypassing the IAP congestion. As a result of significance of these procedures in the opposition to anti tumefaction therapies, several structural PF 573228 and functional studies on Bax have already been published. It is obvious that many different, usually hardly suitable email address details are reported. Several facets contribute to this situation, including the complex structure of proteins interacting with Bax, the different forms of initial, and the different functions that contribute to apoptosis. Many respected reports will be essential to reveal the Bax governed signaling network. As why in some instances Bax service was caspase dependent, although Endosymbiotic theory in other situations it was stopped by caspase inhibition: next, the extrinsic and intrinsic apoptotic signal transduction pathways were practically divided an example, previously, it was long debated. The reply to this question became obvious, meaning that in the intrinsic pathway, Bax is stimulated in a caspase separate manner, whereas caspase 8 is essential for recruiting Bax from the extrinsic pathway. Moreover, we expect that other apparent paradoxes could be solved by increasing the information about the things of Bax initial. Likely, we assume that the multiple alternative paths of Bax service might be individually explained, and associated with an alternative outcome. Most mechanistic studies have focused on t Bid because the trigger, and cytochrome c while the outcome of Bax activation. Ergo, several important questions remain: what Dizocilpine MK 801 is the part of the different Bax domains in the various mechanisms of Bax recruiting Also, the different forms of proteins released from mitochondria stay to be further investigated. Necroptosis, also known as type III programmed cell death, is a standard cell death pathway identified by Degterev et al.. Necrostatin 1. targeting serine?threonine kinase receptor interacting protein 1. Is really a specific inhibitor of necroptosis which will be determined by RIP1/3 complex service. Necroptosis handles the chronic intestinal inflammation, T cell proliferation and normal embryonic growth. Kind II programmed cell death, autophagy, plays an essential role in destruction and recycling cellular components. During vitamins or growth factor an important role is autophagy played by withdrawal; for maintaining cell survival. But, abnormal autophagy can lead to cell death, termed autophagic cell death. Macroautophagy is themost active formof autophagy and in this technique, organelles and cytosolic macromolecules are sequestered into double membrane structures referred to as autophagosomes, which are subsequently sent to the lysosome for degradation.
The refinement of ATM was based on the technique of Goodarzi and Lees Miller. All cells lines were grown at 37 C in five full minutes CO2 in Dulbeccos modified Eagle medium supplemented with 10 percent fetal bovine serum, 100 U/ml penicillin, and 100_g/ml streptomycin. Medium for both GM16666 and GM16667 furthermore included 100_g/ml hygromycin to Bicalutamide molecular weight keep stable cell line selection. Cells grown to 80% confluency in 250mm2 tissue culture flasks were washed 3 x with 20 ml of ice cold hypotonic buffer, obtained employing a cell lifter and centrifuged at 1850 g for 10 min. Cells were resuspended in five times the pellet volume of hypotonic buffer and incubated for 30min at 4 C. Cells were then collected by centrifugation at 1850 g for 30 min and intact nuclei were produced using a homogenizer using a loose fitting pestle. Subsequent focus by centrifugation at 3300 g for 30 min, nuclei were resuspended Plastid in one half the packed nuclear volumeof resuspension buffer. Nuclear lysis stream comparable to one half the stuffed nuclear volume was then added. Nuclei were incubated for 30min at 4 C and afflicted by three rounds of snap freezing in liquid nitrogen and rapid thawing at 37 C. After lysis by Dounce homogenization, nuclear lysates were centrifuged at 25,000 g for 30 min and the supernatantwas dialyzed for 18h at 4 C against dialysis buffer. Aliquots of the trials were snap frozen in liquid nitrogen and stored at 80 C. The protein concentration of the nuclear components was based on the Bradford protein assay utilizing the Bradford reagent and BSA as a standard. HeLa cells were grown to log phase and collected by sedimentation at 10,000 g for 15 min at 4 C. The resulting cell natural compound library pellet was washed twice with 10 ml low salt buffer. The cells were resuspended and collected in 7ml of high salt buffer. This barrier and all future buffers were supplemented with the protease inhibitors PMSF, leupeptin and pepstatin. After disruption employing a Dounce homogenizer, the lysate was centrifuged at 10,000 g for 30 min and the supernatant was saved. The pellet was extracted with 3ml of high salt buffer and centrifuged creating an additional supernatant. S1 and S2 were combined and straight away diluted with TB load to a final conductivity add up to 75mM KCl. P10 was applied onto a DEAE Sepharose quick flow column equilibrated in TB?75mM KCl at an interest rate of 2ml/min. Bound protein including ATM was eluted with 5 column volumes of TB?200mM KCl, following the column was washed with 10 column volumes of TB?75mM KCl. The eluted protein was pooled, quickly diluted to a conductivity add up to 75mM KCl, and put on a ml SP Sepharose fast flow column. Again the column was washed with 10 column volumes of TB?75mM KCl, and eluted with 5 column volumes of TB?200mM KCl.
MTT reduction Bicalutamide Cosudex was evaluated by measuring absorption at 570 nm on a reader and corrected for background absorbance at 630 nm. Each treatment was completed in triplicate and as percentages of untreated cells values are expressed. Tremendously growing VA13, AT22, and EA. hy926 cells were plated at a density of 1. 5?? 103 cells/100 mm tissue culture plate in the absence or existence of lipoproteins in normal growth medium. The cells were preincubated with ATM I for 1 h before addition of lipoproteins, when indicated. After 18 h of incubation, the plates were washed three times with PBS the method was changed, and the cells were cultured for 12 more days. The cells were fixed for 5 min with methanol and stained with crystal violet and cell clusters of 50 or even more cells were counted as colonies under a microscope. VA13 and AT22 cells were seeded in 6 well plates until 50% confluence was reached. After over night serum misery, cells were incubated with indicated concentrations of lipoproteins. At the indicated moments, the cells were trypsinized Retroperitoneal lymph node dissection, washed with PBS and solved in serum free DMEM. The cell suspension was mixed with 1:1 with 0. Four weeks Trypan blue stain. Feasible cells, determined by a clear cytoplasm, were measured using CountessTM mobile counting chamber slides and the CountessTM Automated Cell Counter. VA13 and AT22 cells were seeded in 6 well plates on glass cover slips and cultured in normal growth medium. When cells reached 50% confluence, cells were serum starved over night and incubated with 100 _g/ml lipoprotein for 16 h. Cells were washed with PBS and fixed with 3 months methanol for 5 min. Staining of nuclei was performed with 0. 5 _g/ml bisbenzimide. Cells on glass cover slips were incubated with the fluorescence dye for 30 min in the angiogenic inhibitor dark, cleaned with aqua dest. , air dried and mounted with glycerol. Micronuclei were scored and cell images recorded having an FSX100 Box Type Fluorescence Imaging Device. Before scoring the micronuclei, all slides were coded and randomised. The amount of micronuclei was based on counting 500 cells/slide. The criteria for scoring micronuclei were adapted from sources ; each treatment was done in triplicate. As percentages of how many micronuclei in untreated cells values are expressed. Logarithmically growing VA13 and AT22 cells were plated in 100 mm tissue culture dishes. Once the cells achieved 50% confluence, they were treated with 30 _g/ml lipoproteins for 8 h. Colcemid was added for 4 h, to arrest the cells in metaphase. The cells were washed with PBS and trypsinized. The reaction was stopped with DMEM and cells were pelleted 5 min at 500?? g. Then, the cells were resuspended in 0. 075 mM KCl and incubated for 15 min at 37 C. 2 hundred microliter of Carnoys fixative was added; cells were carefully mixed and pelleted.
we examine recent developments in the discovery of JNK inhibitors and their potential in treating human disease. We first concentrate on supplier AG-1478 small particle, ATP aggressive JNK inhibitors as summarised in. Our preliminary discussion centers on SP600125 manufactured by Signal Pharmaceuticals/Celgene. Furthermore, we offer a quick overview of an ever-increasing quantity of other small particle ATP aggressive JNK inhibitors now defined in the published literature. The recent advances are then discussed by us in the utilization of ATP non competitive JNK inhibitory peptides. These inhibitors are also featured in. Lastly, we consider questions that arise with the growth of JNK inhibitors and their possible therapeutic application. These questions centre on the settings had a need to establish nature of measures of JNK inhibitors, whether JNK isoformselective inhibitors are probable or desirable, Papillary thyroid cancer whether other substances have off goal effects to prevent JNK, and what issues accompany the serious use of JNK specific inhibitors. Further work will be needed seriously to address these dilemmas, however the proven effectiveness of the existing generation of JNK inhibitors in improving outcomes in illness models implies that this further work will pay dividends. In late 2001, the little particle JNK chemical, SP600125 one, was reported following a testing of a library for inhibitors of JNK2 action towards the d Jun transactivation domain. The chemical structure of SP600125 is found in, along with the buildings of other small molecule inhibitors of JNK discussed in subsequent chapters of this review. The extremely planar nature of SP600125 and poor solubility in aqueous solution, both consequences of its anthrapyrazolone core structure, were known in its original description. JNK inhibition by SP600125 was more Bicalutamide Androgen Receptor inhibitor observed to be reversible and ATP aggressive, demonstrating IC50 values for JNK inhibition in the product range of 40?90 nM with N300 fold selectivity over the related mitogen activated protein kinases, ERK1 and p38 2 and between 10 fold and 100 fold selectivity over another 14 protein kinases tested. These results suggested high affinity and specific interactions of SP600125 with elements in the JNK ATP binding site. These relationships of SP600125 with JNK have now been further explored following the co crystallisation of SP600125 with JNK3. The resulting design : 1PMV is shown in, where the JNK3 elements not preserved in the related MAPK, p38 2, have been highlighted. These remains produce a thin ATP binding pocket in JNK that met the planar SP600125 molecule and were predicted to donate to the specificity of SP600125 towards JNK on the p38 MAPKs.