es of the HIV lifecycle. A growing body of work has also implicated CXCR4 and various chemokines in regulating infection of resting CD4+ T cells, an important latent reservoir of HIV in infected individuals. Therefore, it’s plausible that CXCL12responsive phosphoproteins from our study may regulate entry and integration of resting CD4+ T cells. Future studies can also address potential differences between signaling of CXCR4 and CCR5, another HIV-1 coreceptor, since differential engagement of these chemokine receptors can have unique effects on target gene expression and host factor requirements for infection of primary cells. In all, our study uncovered several members of signal 1339058-04-6 transduction pathways that HIV-1 may modulate in order to 
successfully infect T cells, of which targeting has become an attractive avenue for anti-HIV-1 therapeutics. O’Hayre et al. recently published a study examining the phosphoproteome of 14726663” CXCL12-treated primary chronic lymphocytic leukemia cells, a cancer of B cells. Our studies differ in target cell, phosphopeptide enrichment strategy, and method of quantification. Of the 13 phosphoproteins reported by O’Hayre et al. to have spectral counts suggestive of CXCL12-responsiveness, only half were detected as SILAC pairs by at least one phosphopeptide in our analysis, yet none were considered CXCL12-responsive. Interestingly, both of our studies detected novel CXCL12responsive AKT substrates, PDCD4 and AKT1S1 , underscoring the potential role of AKT signaling and leukemias. Phosphoproteomic examination of signaling pathways is poised to greatly advance signal transduction research in areas such as basic science, clinical therapeutics and perhaps even drug design. Our study greatly expands the breadth and diversity of early changes in the CXCL12/CXCR4 signaling network. We have shown through multiple independent means of validation that our dataset is consistent with what is currently understood about CXCL12/CXCR4 signaling. We confirmed associations with various signaling pathways that have already been described, e.g. T cell activation, EGF, and mTOR, and highlighted perhaps under-appreciated associations such as with microtubule dynamics. Our study also uncovered several phosphoproteins that may regulate cancer metastasis and HIV-1 infection of T cells, providing new avenues to expanding not only our basic understandings of these diseases but also to identify novel therapeutics. Materials and Methods CXCL12 treatment of CEM cells CCRF-CEM cells were cultured in RPMI1640 with ” 10% FBS, 100 units/mL of penicillin and 100 mg/ mL streptomycin. For SILAC labeling, cells were cultured in RPMI lacking lysine or arginine supplemented with 200 mg/L 13C6, 15N4 arginine, 40 mg/L 13C6, 15N2 lysine and 10% dialyzed FBS for eight doublings. For proteomic experiments, cells were serum-starved overnight at a density of 16106 cells/mL in SILAC media. CXCL12 stimulations were done in fresh serum-free SILAC media at 56106 cells/ mL for 5 min. Cell suspensions were mixed with ice-cold PBS containing 16 phosphatase inhibitors and centrifuged at 1500 RPM for 5 min at 4uC. Pellets were frozen on dry ice. Protein hydrolysis Cell pellets were removed from storage at 280uC and placed on ice. Lysis buffer with protease inhibitors was used to disrupt the cell pellet and heated at 90uC for 5 min. The sample was incubated with 5 Units of benzonase for 10 min at room temperature. Cysteines were alkylated by the addition of 50 mM iodoacetamide