To further establish the green fluorescence intensity of GFPBclxL was lowered by DsRed and its plan DsRed Express2, we reviewed green fluorescence of cells by flow cytometry. The common green fluorescence intensity was demonstrably diminished by overexpression of DsRed and DsRed Express2. The normalized green fluorescence intensity was lowered to 22. 4-5 and 30. 4-6, respectively. We then carried out western blotting to analyze the protein expression degree of GFP Bcl xL. The results showed that the quantity of GFP Bcl xL protein is significantly lower in cells expressing DsRed and GFPBclxL than that in cells expressing GFP Bcl xL only. Similar effects were also obtained Letrozole CGS 20267 in cells expressing DsRed Express2 and GFP Bcl xL. Further, we discovered that the endogenous Bcl xL protein levels were also lowered in HeLa cells co transfected with plasmids encoding DsRed or DsRedExpress2 with GFP Bcl xL. Consequently, the over expression of DsRed o-r DsRed Express2 may result in endogenous exogenous and BclxL GFP Bcl xL protein levels, which explains the lowered green fluorescence intensity in HeLa cells. To decrease the GFP Bcl xL protein degree, DsRed could act to increase the protein degradation, or down regulate both the protein or the mRNA production. To recognize these possibilities, we built a encoding GFP Bcl xL, in so that only GFP protein might be made although the mRNA contained Cellular differentiation the coding sequence of Bcl xL which a stop codon was introduced between Bcl and GFP xL coding sequences. Curiously, when plasmids coding DsRed and GFP Bcl xL were corp transfected in-to HeLa cells, the green fluorescence intensity was nevertheless weaker than that of cells showing DsRed and GFP. Similar results were also observed in cells expressing GFP Bcl xL and DsRed Express2. Considering if you have no Bcl xL code string that DsRed or DsRed Express2 doesn’t influence GFP protein production, these effects suggest that DsRed or DsRed Express2 represses expression of Bcl xL by transcription or translational regulation. We next investigated whether DsRed inhibited the transcription of Bcl xL in HeLa PF 573228 cells. We extracted the sum total RNA of HeLa cells cotransfected with plasmids encoding GFP Bcl xL and DsRed or empty vector, and used RT PCR to amplify a of GFPBclxL cDNA. As shown in Fig. 4A, DsRed didn’t prevent the transcription of GFP Bcl xL mRNA, since the band intensities of RT PCR products are similar. We then transfected plasmids coding DsRed o-r empty vector in-to HeLa cells, and examined whether the transcription of endogenous Bcl xL was suffering from the overexpression of DsRed. Identical to exogenous effects, DsRed also did not prevent the transcription of endogenous Bcl xL.