Anti na k atpase

Protein samples were run in 8% acrylamide (BioRad) gels or 4–12% precast gradient gels (Invitrogen). Electrical transfer to PVDF membranes (BioRad) was performed in a standard 25 mM Tris, 192 mM glycine, pH~8.3 buffer supplemented with 0.002% SDS for 90 min at 90 V, at 4°C. Membranes were handled in PBS containing 0.2% Tween-20 (PBST). Blocking buffer was 10% newborn calf serum (NCS, Gibco) plus 1% fish gelatin (Sigma) in PBST. Primary antibodies were used at 1:750–1:1000 dilutions, and were purchased from Sigma (anti-L_II–III and anti-Nt), Cell Signaling (anti-PARP), and Abcam (anti-Na⁺/K⁺-ATPase). Anti-Ct, generated against residues 2155–2171 of the rabbit cardiac Ca_v1.2 (Hulme et al., 2006 (link)), was a courteous gift from Drs. W.A. Catterall and R. Westenbroek (University of Washington, Seattle). Secondary antibody (goat anti-rabbit, HRP-conjugated, SantaCruz Biotechnologies) was used at 1:2000 dilution. Protein bands were visualized using enhanced chemiluminescence reagents (Pierce) on X-ray film (Kodak). For stripping and reprobing of PVDF membranes, the stripping buffer was 100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8. Coomassie Blue staining was performed using Biosafe Coomassie (BioRad) according to the manufacturer’s instructions, or using a stain consisting of 0.1% Coomassie Brilliant Blue R-250 in 40% MeOH and 1% acetic acid.