Anti mouse and anti rabbit igg horseradish peroxidase hrp

Cells were collected, washed twice with Phosphate Buffer Saline (PBS), and stored at −80°C until processed. For total protein extraction, cells were lysed with a lysis buffer (150 mM NaCl, 1% Triton X-100, 0.4 mM NaF, 0.4 mM NaVO₄, 25 mM Tris-HCl, pH 7.6 and 1× protease inhibitor) for 45 min on ice and then centrifuged for 10 min at 4°C. The supernatants were collected for further analysis. Protein concentration was determined using Bio-Rad Protein Reagents (Bio-Rad, Hercules, CA). Protein lysates (50 μg) were separated by SDS-PAGE, blotted onto membranes, and probed with the appropriate dilution of each primary antibody. Membranes were rinsed and incubated with a horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using enhanced chemiluminescence (ECL) regents (GE Healthcare, Piscataway, NJ) and autoradiography using a FluorChem^™ 8900 (Alpha Innotech Corporation, San Leandro, CA). The primary antibodies used were: anti-RBPMS (24 kDa), anti-RCBTB1 (58 kDa), anti-ZNF608 (162 kDa) (Novus Biologicals, Littleton, CO), and anti-β-actin (42 kDa) (Sigma, St. Louis, MO). The secondary antibodies used were anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP) (Cell Signaling, Beverly, MA).