Supplementary Figure 7: Themis2 is critical for B cell activation with soluble HEL and associates with PLCγ2 independently of Grb2 binding.
From: Themis2 lowers the threshold for B cell activation during positive selection
(a) Mean B cell phosphospecific antibody binding to p38 and pAKT, after stimulation of mixed Themis2-/- and wild-type IgHEL B cells for 5 min with sHEL. (b) pERK activation following Themis2-/- and wild-type IgHEL B cell stimulation with indicated concentrations of anti-IgM F(ab’)2, showing a dose-response curve following 5 min incubations (top) and time-course (bottom). (c) Time course of mean phospho-specific antibody binding to pPLCγ2, after stimulation of Themis2-/- and wild-type IgHEL B cells with 100ng/ml sHEL or 10μg/ml anti-IgM F(ab’)2. In all experiments, MACS sorted IgHEL CD45.1 Themis2-/- and CD45.2 WT IgHEL B cells were co-cultured in triplicate and mean fluorescence levels evaluated by flow cytometry. Symbols show means (wild-type filled circles and Themis2-/- open circles) with bars 95% confidence limits and unpaired t tests *P<0.05 and **P <0.01. Each graph is representative of at least 3 independent experiments. (d) Anti-Myc and anti-FLAG stained western blot (WB) of crude extract and anti-FLAG immunoprecipitates (IPs) from HEK 293 cells expressing combinations of Myc-tagged Grb2, Flag-tagged Themis2 and FLAG-tagged Themis2-dPRR, which lacks the Grb2 binding site. (e) Anti-Themis2 and anti-FLAG stained WB of anti-Themis2 and anti-PLCγ2 IP from HEK 293 cells expressing combinations of Flag-tagged PLCγ2, Flag-tagged Themis2, and Flag-tagged Themis2-dPRR.
