Co-immunoprecipitation of HA-BCA3 Complexes

Co-immunoprecipitation was carried out as previously described [12 (link),17 (link)]. Briefly, HEK-293 cells were grown on 100 mm plates and transfected with HA-BCA3 or HA-Δ6BCA3. At 48 h post-transfection, the cells were washed with PBS and lysed in 500 µL CO-IP buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM MgCl₂, 0.5% NP-40) containing Halt Protease Inhibitor Mix (Thermo Scientific) for 30 min on ice. One-fifth of the total cell lysate sample was used for Western blot analysis. The rest of the cell lysate was cleared by centrifugation and diluted with 1 mL CO-IP A buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl₂). The primary antibody, monoclonal anti-PKA(C) or anti-HA, was added to the cell extract and incubated overnight at 4 °C. The next day, 20 µL Protein-A/G-Sepharose beads were added, and after 2 h incubation at 4 °C, the immunocomplexes were collected by centrifugation. The pellets were washed with CO-IP A buffer and PBS, and immunoprecipitated proteins were separated by SDS–PAGE, transferred onto a nitrocellulose membrane, and detected by Western blot analysis.