PKP2 regulates PKC signaling. (A) Enhanced PKC substrate phosphorylation during PKP2 knockdown. SCC9 cell lysates from nontargeting (NT) or PKP2 siRNA-transfected cells or cells treated with DMSO, 15 nM PMA, or 12.5 μM BIM for 30 min were probed for PKC substrates phospho-MARCKS or -adducin, total MARCKS, adducin, PKP2, or α-tubulin (loading control). Densitometry numbers below the blots represent ratios of phosphoprotein normalized to total protein from treatment samples relative to the NT siRNA sample. (B) PKCα coimmunoprecipitates the PKP2 N-terminal head but not the central armadillo repeat domain. FLAG-tagged PKCα and myc-tagged PKP2-Head or -Arm were cotransfected into HEK293 cells and subjected to anti-FLAG IP. Blots were probed with anti-myc or anti-FLAG antibodies. (bottom) A schematic of PKP2 constructs. 5% input blots represent 5% of triton lysate from which IP was performed, removed before IP for immunoblot analysis. (C) PKCα coimmunoprecipitates endogenous DP in HEK293 cells. FLAG-tagged wild-type PKCα or myristylated (constitutively active) PKCα were transfected into HEK293 cells and subjected to FLAG IP. Precipitates and lysates were probed for endogenous DP or FLAG. Data are representative of three independent experiments. (D) PKP2 is required for PKC–DP interaction. PKCα-FLAG was immunoprecipitated from HEK293 cells transfected with PKP2 or NT siRNA. Endogenous DP, PKP2, and FLAG were probed. Data are representative of three independent experiments. Densitometry (right) reveals 95% reduction in DP coimmunoprecipitated with PKCα.