Distinct PKC isoforms phosphorylate IBtkγ. (A) GST-IBtkγ (1 μg) was in vitro phosphorylated with purified PKCmix (25 ng), recombinant PKCβ (50 ng), or PKCμ (50 ng) in the presence of [γ-³²P]ATP and analyzed by 4%–12% NuPAGE followed by autoradiography and Coomassie blue staining. (B) Schematic representation of GST-IBtkγ constructs with putative PKC phosphorylation sites. (C) GST or GST fused with N-terminus (aa 30-130) or C-terminus (aa 131-240) of IBtkγ (5 μg) were incubated with PKCmix (100 ng) in the presence of [γ-³²P]ATP; proteins were analyzed by 4%–12% NuPAGE followed by autoradiography and Coomassie blue staining. (D) HEK 293T cells (1 × 10⁶) were transfected with expression vectors of PKC isoforms or empty vector (1.5 μg) together with pCMV7.13xFLAG-IBtkγ or the relative empty vector (2 μg); 48 hours after transfection, cells were treated for 45 minutes with PMA, and lysates were analyzed by 10% SDS-PAGE followed by Western blotting with anti-phPKC substrate and anti-FLAG antibodies.