Fig 2: Identification of specific SH2 substrates in PV stimulated cells (A) CD79a, CD22, and FcγRIIB are known tyrosine phosphorylated receptors that recruit SH2 domains. (B) (Top) Lysates of splenic T cells activated for 72 h with anti-CD3/CD28 were probed with SH2 domains from indicated molecules. PD1 is enriched specifically by the SHP2(SH2)2 and SHIPSH2. (Bottom) Quantification of PD1 staining normalized to the bead control. Data is plotted from 3 independent experiments. (C, D) Cells were stimulated with PV prior to treatment with N-glycanase. FcγRIIB and PD1 have observed molecular weight shifts post N-glycanase treatment in WCL, SH2 reagent, and immunoprecipitation lanes. Bars in (B) represent ± SEM; immunoblots are representative of n = 3 independent experiments.

Fig 3: Depleting GMFγ does not impair CD79 phosphorylation induced by soluble anti-Ig antibodies. Cells were transfected with either control siRNA or GMFγ siRNA. (A) Raji D1.3 B cells were stimulated with 20 μg/mL goat anti-mouse IgM to initiate signaling through the D1.3 BCR. (B) Ramos B cells were stimulated with donkey anti-human IgM. Cell lysates were analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79a ITAM (pCD79a), actin (loading control), or GMFγ. The left panels show a representative experiment. The right panels show results from 4 independent experiments. For each sample, the pCD79a band intensity was divided by the corresponding actin band intensity (loading control). The resulting ratios were normalized to that for the 0 min control siRNA cell sample (defined as 1.0) in the same experiment. On the graph, each of the four experiments is indicated by a different symbol. The bars show the mean ± SEM. Paired t-tests were used to calculate p-values. ns, not significant (p > 0.05).

Fig 4: Physiologic stimuli induce phosphorylation of physiologic targets which bind specific SH2 domains. Ex vivo splenic B cells were stimulated with either 10 μg/mL of anti-IgG H+L or 6.4 μg/mL F(ab’)2 anti-IgG H+L per condition. (A) 5E5 cell equivalents of WCL from unstim and anti-IgG H+L or F(ab’)2 anti-H+L were loaded and run by SDS-PAGE and blotted for pFcγRIIB, FcγRIIB, pCD79a, CD79a, and Actin. (B) 10E6 cells were left unstimulated, stimulated with anti-IgG H+L, or F(ab’)2 anti H+L, lysates were subjected to pull down using SHIP1SH2 reagent. Eluted proteins were run with 2E6 cell equivalents of WCL per condition and blotted for pFcγRIIB and total FcγRIIB. (C) 10E6 cells were stimulated with anti-IgG H+L and F(ab’)2 anti-H+L and subjected to pull down using SH2 domain reagents. Adsorbed proteins were eluted, run on SDS-PAGE and blotted for pFcγRIIB and pCD79a. Immunoblots are representative of n = 3 independent experiments.

Fig 5: WAVE2 KD does not reduce the expression of Hem1 protein and does not affect BCR signaling in response to immobilized anti-Ig. (A) A20 cells were transfected with control siRNA or WAVE2 siRNA, cultured for 48 h, and then analyzed via immunoblotting with Abs to WAVE2, Hem1, or actin (loading control). Normalized levels of WAVE2 or Hem1 relative to control siRNA-transfected cells are shown. (B) A20 cells that had been transfected with control siRNA or WAVE2 siRNA were allowed to spread on anti-IgG-coated tissue culture wells for the indicated times before analyzing BCR signaling by immunoblotting for the phosphorylation of the CD79a ITAM (pCD79a) or the phosphorylated (activated) forms of ERK (pERK) or Akt (pAkt). The same cell lysates were probed for total CD79a, ERK, or Akt. The dashed red line was overlaid on the images of the blots to visually separate the time courses for the control siRNA and WAVE2 siRNA-transfected cells. For both panels, one of three independent experiments with similar results is shown. Full uncropped blots are shown in Supplementary Materials Figure S1.