Figure 5. PKCε mediates SMK-induced TACE and EGFR phosphorylation.

Smoke stimulates phosphorylation and activation of TACE. (A) NCIH292 cells were incubated with SFM, smoke containing SFM (SMK), or EGF (10 ng/ml)-containing SFM for 10 min. (A) Cell lysates were separated by electrophoresis and immunoblotted with anti-TACE or anti-isoformspecific PKC antibodies as indicated. Actin was used as protein level loading control. (B) Cell extracts samples were immunoprecipitated (IP) with anti-TACE antibody, and immunoblotted (IB) with anti-isoformspecific PKC or anti-TACE antibodies as indicated. (C) Cell extracts samples were immunoprecipitated (IP) with anti-isoformspecific PKC, and immunoblotted (IB) with anti-TACE or anti-isoformspecific PKC antibodies as indicated. (D) Cultured NCIH292 cells were treated for 10 min with SFM, SMK or PMA (400 nM). Cell lysates were fractionated into a membrane and a cytosolic fractions then separated by electrophoresis and immunoblotted with anti-isoformspecific PKC antibodies as indicated. (E) NCIH292 cells were transfected with isoformspecific PKC siRNAs as indicated prior to stimulation with smoke-containing SFM, and immunoblotted with anti-isoformspecific PKC antibodies. Actin is immunoblotted as a loading control. NCIH292 cells or NCIH292 knockdown for PKCε or PKCα or PKCδ were incubated with SFM, smoke containing SFM (SMK), or EGF (10 ng/ml)-containing SFM for 10 min. Cell extracts samples were either (F) immunoprecipitated (IP) with anti-TACE antibody, and immunoblotted (IB) with anti-phosphoserine or anti-TACE antibodies, or (G) prepared and TACE activity was measured as described in Fig. 1. (Double asterisks indicate significantly different from SFM (p<0.01)), or (H) immunoprecipitated (IP) with anti-EGFR antibody, and immunoblotted (IB) with anti-phosphotyrosine or anti-EGFR antibody, antibodies as indicated.