Fig. 3. PCTAIRE kinases are expressed in many cell lines and can be co-immunoprecipitated with Sec23Ap. (A) Analysis of PCTAIRE expression in cell lysates from four different cell lines: HeLa (cervical epithelia), A549 (lung epithelia), HEK-293 (embryonic kidney), MDCK (Madin-Darby canine kidney). (B) Sec23Ap can be co-immunoprecipitated with PCTAIRE-3 from MDCK cell lysates (lanes 1-3) and PCTAIRE-1 from HeLa cell lysates (lanes 4-6). Immunoprecipitates were washed extensively, separated by SDS-PAGE followed by immunoblotting with anti-Sec23p. Lanes 1 and 4, 5% input lysate; lanes 2 and 5, immunoprecipitation with anti-PCTAIRE-3 (lane 2) or anti-PCTAIRE-1 (lane 5); lanes 3 and 6, control immunoprecipitation using rabbit IgG. ^*, non-specific band at 100 kDa. (C) Specificity of co-immunoprecipitation was confirmed using antibodies directed against CDK1. Unlike anti-PCTAIRE-1, neither mouse IgG nor anti-CDK1 was able to immunoprecipitate Sec23Ap from HeLa cell lysates. (D) In the converse experiments, neither anti-Sec23Ap nor anti-PCTAIRE-1 were able to immunoprecipitate CDK1.