Mouse monoclonal anti actin antibody

Cells were lysed in sodium dodecyl sulfate (SDS) buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue), and the amount of protein in each sample was determined with the bicinchoninic acid assay (Beyotime, Shanghai, China). Ten to twenty micrograms of total cellular protein from each sample were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) and proteins were detected with corresponding primary and secondary antibodies. Blots were performed using an enhanced chemiluminescence detection kit (Thermo Scientific, Inc., Waltham, MA, USA). Primary antibodies were: anti-phospho-Rb (Ser 795) (Sigma-Aldrich, MO, USA), anti-cyclin D1 (Cell Signaling Technology [CST], MA, USA), anti-cyclin E1 (CST, MA, USA), anti-P21 (Santa Cruz Biotechology, CA, USA), anti-P27 (Santa Cruz Biotechology, CA, USA), anti-P53 (CST, MA, USA), anti-PERK/ p-PERK (Abcam, MA, USA), anti- eIF2α/ p- eIF2α, anti-ATF4 and anti-CHOP (CST, MA, USA). A mouse monoclonal antibody against the NDV NP protein was prepared in our laboratory for use in these experiments. Actin was detected with a mouse anti-actin monoclonal antibody (Sigma-Aldrich, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).