The anti phosphoserine antibody was from Abcam Inc. The antibodies used for immunoprecipitation CC-5013 Western blotting were against total mTOR FRAP and total ER from Santa Cruz Biotechnology Inc. Although in Additional file 1 Figure S1 there appears to be a partial reduction in IHC signal due to the non phosphorylated peptide and an irrelevant p peptide, close examination of Additional file 1 Figure S1C and 1D and comparison to Additional file 1 Figure S1A shows that the dynamic signal intensity range amongst positive cells is similar. In Additional file 1 Figure S1D there is a very dark brown staining section between 180 and 270 as well as a small circle of intense staining at 180. in Additional file 1 Figure S1C an intensely staining focus of cells is found at 90 and 135.

These are similar in intensity to regions in Additional file 1 Figure S1A. Even Inhibitors,Modulators,Libraries in Additional file 1 Figure S1A there is a range of staining intensities reflecting the heterogeneous nature of tumors and the cells within them. Each different staining is done on a serial section which Inhibitors,Modulators,Libraries increases the heterogeneity. As well it should be noted that the counter stain in Additional file 1 Figure S1A is generally more intense than Additional file 1 Figure S1C and 1D, which effects the perception of intensity. In contrast all signal is lost in Additional file 1 Figure 1B where the antibody is pre absorbed with excess of the specific phospho peptide. The data seen in these figures support the well known heterogeneity of expression of any protein that seems to occur in breast tumor cells in vivo in a breast biopsy specimen.

So perceived differences we argue are due primarily to tissue composition and tumor cell heterogeneity and not due to a lack of phospho epitope specificity, although we can not completely eliminate this possibility. Tissue collection times As previously described a cohort of breast tumors for which Inhibitors,Modulators,Libraries the collection time, as defined Inhibitors,Modulators,Libraries previously is available in the Manitoba Tumor Bank. This timed collection cohort was used to determine if detection of p mTOR and p p70S6K varied significantly with time of biospecimen collection. Formalin fixed paraffin embedded blocks from 133 cases had sufficient material to be used for this study. IHC for both p mTOR and p p70S6K was carried out on adjacent sections. Within this cohort the collection time ranged from 5 to 276 minutes.

Although there may be a trend for p mTOR IHC score to decrease with time no statistically significant relationship between collection time and p mTOR or p p70S6K was found. The tumors were also divided into groups based on collection times of 30 mins versus Inhibitors,Modulators,Libraries 30 mins. Mann Whitney two tailed analyses showed no significant differences in the IHC score between the two time groups for either p mTOR or p p70S6K as illustrated in Additional file 2 Figure S2. Immunohistochemistry no IHC for TMAs was performed as described previously. Serial sections were stained with antibodies as previously described.