PKC phosphorylation of IBtkγ at serine 90 reduces the affinity binding of IBtkγ to Btk. (A) Aliquots (5 μg) of GST, GST-IBtkγ (aa 30-240), GST-IBtkγ (aa 30-130), or the derivative mutants S90A, S87/90A were in vitro phosphorylated with PKCmix (150 ng) or left untreated, and then incubated with lysates of HEK 293T cells expressing Btk. After GST pull-down, proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-Btk and anti-phPKCsub Abs and Ponceau staining. A representative experiment of 2 independent experiments is shown. (B) DeFew cells (2.4 × 10⁷) were transfected with wild-type FLAG-IBtkγ plasmids or mutants (120 μg) and 24 hours later were stimulated for 10 minutes with anti-IgM or left untreated; cell lysates were immunoprecipitated with anti-FLAG Ab and analyzed by 4%-12% NuPAGE followed by Western blotting anti-Btk and anti-FLAG antibodies. The levels of Btk bound to IBtkγ were measured by densitometry (bottom). A representative experiment of 2 independent experiments is shown.