Mouse or rabbit igg hrp conjugate secondary antibodies

Protein was extracted from cells using RIPA buffer (Sigma-Aldrich) with 1% v/v protease inhibitor cocktail (Sigma-Aldrich), 1% v/v phosphatase inhibitor cocktail 2 (Sigma-Aldrich), 1% v/v phosphatase inhibitor cocktail 3 (Sigma-Aldrich), and 1 mM PMSF (Sigma-Aldrich). Protein lysates were centrifuged at 12,000 rpm at 4°C for 15 minutes to remove cell debris. Total protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific). Following SDS-PAGE, proteins were transferred to PVDF membranes. Membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% milk for 60 minutes at room temperature. The following antibodies were diluted in TBST containing 0.5% milk, and incubated with membrane overnight at 4°C: calpastatin-pS633, calpastatin-pS351 (custom ordered from Genscript), SMC1A, SMC1A-pS957, beta-actin, tubulin, calpastatin, calpain-1, calpain-S1, myc-tag, ATM-pS1982, ATM, Chk2, and Chk2-pT68 (Cell Signaling Technology). Mouse or rabbit IgG-HRP conjugate secondary antibodies (Cell Signaling Technology) and an enzyme-linked chemiluminescence kit (Pierce) were used to detect proteins.