Fig. 3
CaV3.3 channels and β1b-subunit do not coimmunoprecipitate. Western blot of IP of CaV1.2-GFP with β1b-HA (positive control, lane 1) and CaV3.3-GFP with β1b-HA (lanes 2 and 3). IPs in lanes 1 and 2 were washed three times with lysis buffer after overnight incubation with the indicated proteins, whereas the IP from lane 3 was washed only once. The immunoblot was probed with an antibody against GFP (upper panel), then striped and probed with an anti-HA antibody (middle panel), then striped again and probed with a homemade anti-actin antibody (lower panel). Lanes 4-6 were loaded with 15 µg of total protein from lysates of HEK-293 cells transfected with β1b-HA, CaV1.2-GFP and CaV3.3-GFP, respectively. Finally, lanes 7 and 8 were loaded with 10 µl of the IP supernatant from lanes 1 and 2, accordingly. Notice the importance of exhaustive washing procedures, since incomplete washing of the beads could lead to false positives results. As can be observed, CaV1.2 channels coimmunoprecipitate with β1b-HA (lane 1, upper panel), whereas CaV3.3 channels do not (lane 2, upper panel). Representative figures of three independent experiments