ith similar studies . Only when a heavy and light peptide pair within a given sample is detected by the mass spectrometer can the CID-25010775 relative abundance of a phosphopeptide be determined. About 1,200 SILAC pairs were detected in each experiment, with an average 40.863.5% shared between any pair of experimental samples. There was no statistical difference between the average overlaps of technical replicate and biological replicate comparisons. From all four replicates, 4,074 unique SILAC pairs were identified from 1,673 proteins. CXCL12-regulated phosphoproteome We established two criteria to identify phosphopeptides that were potentially regulated by CXCL12. First, the change in phosphopeptide abundance upon CXCL12 addition must be increased or decreased by $1.5-fold. Second, a phosphopeptide must be consistently regulated in two or more biological replicates – the HS 
sample and any two of the LS1a, LS1b or LS2 samples. 14726663” While both heavy and light stimulations have not been routinely included in published quantitative phosphoproteomics studies, this criterion reduces potential false positives resulting merely from CXCL12-independent differences in peptide abundances between the heavy and light cells. We reasoned that phosphosites that are strongly regulated by CXCL12 would be detected in biological replicates regardless of potential variations due to heavy and light media preparations or due to biological variability. Compared to unphosphorylated peptides, 11.062.5 times more phosphopeptides increased in abundance $1.5fold upon CXCL12 treatment, indicating a good degree of specificity. This is consistent with the fact that cells were treated with CXCL12 for only 5 min – enough time for changes in phosphorylation via CXCR4-dependent signaling, but not enough time for many proteins to change in overall abundance due to either degradation or enhanced protein synthesis. Ratios of protein abundance can also be derived through quantification of several unphosphorylated peptides from the same protein. Using this approach, we found that only one out of 3,187 proteins consistently changed in abundance more than 1.5-fold in biological replicates. In contrast, 17062696” 89 phosphopeptides from 81 proteins consistently changed in abundance by at least 1.5-fold between any pair of biological replicates. Mass spectrometry details of these phosphopeptides are included in Phosphoproteomics of CXCL12 Signaling CXCL12 and so may have had unequal abundances in the heavy and light cell populations. Validation of CXCL12-responsive phosphosites To validate the CXCL12-responsive phosphosites, we compared them to known CXCL12-responsive phosphosites and also tested novel ones with phosphospecific antibodies by Western blot. About 50 phosphosites have been shown to be regulated by CXCL12 at different times in diverse cell types. Since we examined only a single time point in a single cell type, only a subset of these phosphosites would likely be detected in our study. Indeed, eight of these phosphopeptides were detected as SILAC pairs in biological replicates. AKT1, ERK2, GSK3B and RSK1, all known CXCL12-responsive phosphosites, surpassed the 1.5-fold change in biological replicates. In addition, the autophosphorylation site of PAK2 and the homologous site in PAK4, which correlate with its kinase activity, also increased upon CXCL12 addition. Two other phosphosites previously shown to be regulated by CXCL12, RPS6, increased 1.5- fold, but in only one biological replicate and s
