Fig. 2. Co-immunoprecipitation of endogenous PKB and periplakin. (A) Characterization of generated anti-periplakin antibodies. Purified GST and GST-c-ppl were separated by SDS-PAGE and immunoblotted with anti-GST or anti-GST-c-ppl (5117). (B) Cell lysates of COS-7 cells transfected with myc-c-ppl were either immunoprecipitated with anti-GST-c-ppl (5117 and 5118), anti-periplakin peptide serum (7445 and 7446; left panel), or anti-myc (9E10; two right panels). Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-myc (9E10; right panel), anti-periplakin peptide serum (7445) and anti-GST-c-ppl (5117; two left panels). (C) MCF-7 cells were lysed and periplakin was immunoprecipitated with anti-GST-c-ppl or anti-peptide serum. (NI) is an immunoprecipitation with non-immune serum as a control. Immunoprecipitates were separated by SDS-PAGE and immunoblotted for the presence of periplakin with 5117. (D) Epithelial cells express periplakin. Various cell lines were lysed and analyzed by western blotting using the anti-periplakin 5117 antibody. (E) Periplakin interacts with PKB endogenously. Periplakin and PKB were immunoprecipitated from MCF-7 cells using the anti-periplakin 5117 antibody and anti-PKB antibody, respectively, and the immune complex was resolved by SDS-PAGE and immunoblotted with the anti-periplakin 5117 antibody. Non-immune anti-serum was used as a negative control. (F) c-ppl expression does not affect insulin-induced PKB activation. A14 cells were transfected with HA-PKB either in the presence or absence of myc-c-ppl. After 36 hours cells were either treated with insulin for 7 minutes or left untreated. HA-PKB was immunoprecipitated and analysed for kinase activity as described (Burgering and Coffer, 1995).