Figure 2

Co-immunoprecipitations of GPKOW and PKA Cβ2. A. 293T cells were transfected with native PKA Cβ2 and GPKOW with a C-terminal V5-tag (lanes 1-3) or with native GPKOW and PKA Cβ2 with a C-terminal V5-tag (lanes 4-6). Twenty hours post transfection the cells were harvested and lysed. The lysates were adjusted to equal protein concentration for each experiment and precleared with magnetic beads before input samples were collected (lanes 1 and 4). The lysates were subjected to immunoprecipitations using anti-PKA Cβ2 (lane 3), anti-GPKOW (lane 6) or rabbit IgG (lanes 2 and 5) and magnetic beads. All samples were analyzed by SDS-PAGE and immunoblotting using anti-PKA C (lower panel lanes 1-3), anti-GPKOW (lower panel lanes 4-6) or anti-V5 (upper panel). One representative experiment clearly showing interaction is presented. B. 293T cells were co-transfected with native GPKOW and PKA Cα1 (lanes 1-3), PKA Cβ1 (lanes 4-6) or PKA Cβ4 (lanes 7-9) and treated as described in A. Input samples (lane1, 4 and 7) and immunoprecipitations with anti-GPKOW (lane 3, 6 and 9) or rabbit IgG (lanes 2, 5 and 8) were immunoblotted with anti-GPKOW (lower panel) or anti-PKA C (upper panel). One representative experiment is presented. C. 293T cells were co-transfected with PKA Cβ1 and PKA RIα, treated as described in A and immunoprecipitated with anti-PKA RIα (lanes 2-5). One pellet were treated with the cAMP analog, 8-CPT (lanes 4-5), and all pellets (P) and supernatants (S) were analyzed by SDS-PAGE and immunoblotting using anti-PKA C (upper panel) or anti-PKA RIα (lower panel). One representative experiment is shown.