Western blot

Transiently transfected HEK293 cells grown on 35 mm diameter tissue culture dishes were harvested, suspended in lysis buffer (50 mm Tris‐HCl, pH 7.40, 150 mm NaCl, 0.4% deoxycholate and 1% Triton X‐100 supplemented with 2 mm phenylmethylsulphonyl fluoride, 12 μg ml⁻¹ aprotinin, 1 μm benzamidin, 21.5 μm leupeptin, 5 μm pepstatin A and calpain inhibitors from Sigma) and sonicated 48 h after transfection. Then, 50 μg of input protein was resolved by SDS‐PAGE and transferred to 0.2 μm polyvinylidene fluoride membrane. To detect multiple proteins, blots were cut and incubated with anti‐Cav3 (catalogue number 610421; Becton‐Dickinson Biosciences, Franklin Lakes, NJ, USA) or anti‐GAPDH (MAB374; Millipore Sigma, Burlington, MA, USA) and then incubated with HRP‐conjugated secondary antibodies.