Cells were lysed in 500 μl co-immunoprecipitation assay (co-IP) lysis buffer (Thermo Scientific) on ice for 30 min. The protein extracts were incubated with anti-IgG, -Smad2, -Smad3, or -Smad4 antibodies at 4°C for 2 h, followed by the addition of 10 μl protein A/G-agarose beads (Santa Cruz Biotechnology) and incubation at 4°C overnight. Immunoprecipitates were washed three times with RIPA buffer and then resuspended and boiled in SDS loading buffer. Western blotting was performed using an anti-RGC-32 antibody generated by us [19 (link)].