Anti k8.1 mouse monoclonal

Cells were lysed in immunoprecipitation lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing protease inhibitors (Roche, Basel, Switzerland). Total cell lysates (25 μg) were boiled in SDS-PAGE loading buffer, subjected to SDS-PAGE, and subsequently transferred to a polyvinylidene fluoride membrane (Millipore-Sigma, St. Louis, MO, USA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA). The final dilution of the primary antibody was 1:5,000 for anti-K-Rta rabbit serum, 1 μg/ml anti-K8α (Santa Cruz, Santa Cruz, CA, USA), 1 μg/ml anti-ORF57 mouse monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA) and anti-LANA rat monoclonal (Millipore-Sigma, St. Louis, MO, USA), 1 μg/ml anti-K8.1 mouse monoclonal (Santa Cruz, Santa Cruz, CA, USA), 1:1,000 anti-RFP monoclonal antibody (Thermo Fisher), 1:500 anti-ORF52 monoclonal antibody, and 1:5,000 anti-β-actin mouse monoclonal (Millipore-Sigma, St. Louis, MO, USA). Washing membranes and secondary antibody incubations were performed as described previously (50 (link)).

Cells were lysed in IP lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing protease inhibitors (Roche, Basel, Switzerland). Total cell lysates (25 µg) were boiled in SDS-PAGE loading buffer, subjected to SDS-PAGE, and subsequently transferred to a polyvinylidene fluoride membrane (Millipore-Sigma, St. Louis, MO) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Streptavidin-HRP conjugate was used at a 1:3,000 dilution. The final dilutions or concentrations of the primary antibodies were 1:5,000 for anti-K-Rta rabbit serum, 1 µg/ml for anti-K8α (Santa Cruz, Santa Cruz, CA), 1 µg/ml for anti-ORF57 mouse monoclonal antibody (Santa Cruz), 1 µg/ml of anti-K8.1 mouse monoclonal (Santa Cruz), and 1:5,000 for anti-β-actin mouse monoclonal antibody (Millipore-Sigma, St. Louis, MO). Membrane washing and secondary antibody incubations were performed as described previously (58 (link)).