Fig. 2.
AKAP15 associates with the distal C terminus of CaV1.2 via LZ-like motifs in tsA-201 cells. (A) Lysates of tsA-201 cells transfected with CaV1.2 and AKAP15 were immunoprecipitated with anti-CNC1 (lane 2) or control IgG (lane 3). Immunoblots were probed with anti-CNC1 (Upper) or anti-AKAP15 (Lower). Positive control for immunoblotting was 20 μl of lysate (lane 1). (B) TsA-201 cells were cotransfected with wtCaV1.2 ABD and wtAKAP15 (lanes 1), wtCaV1.2 ABD and AKAP15LZm (lanes 2), CaV1.2 ABDLZm and wtAKAP15 (lanes 3), or CaV1.2 ABDLZm and AKAP15LZm (lanes 4). Cell lysates were immunoprecipitated with an anti-myc antibody or nonimmune IgG, and the immunoprecipitated AKAP15 protein was detected using the RII-biotin assay (Upper). Immunoblotting of cell lysates with RII-biotin (Lower Left) or an anti-myc antibody (Lower Right) shows equivalent expression of the AKAP15 and CaV1.2 ABD protein, respectively. (C) Immunoblot of tsA-201 cell lysate expressing CaV1.2 was probed with chicken anti-CNC1 to verify that this antibody specifically recognizes CaV1.2 channel protein.