Western Blot Protein Analysis Protocol

Cells were washed with PBS, lysed in lysis buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol) and boiled for 3min. The protein concentrations of the lysates were quantified with a BCA Protein Assay Kit (Thermo Fisher). Protein samples were separated by SDS-PAGE using 10% agarose gel and transferred to transfer membranes (Millipore-Sigma, St. Louis, MO, USA), which were incubated in 5% nonfat milk (A600669, Sangon) at room temperature for 2 hours. The membrane was incubated with the primary antibody at 4°C overnight or at room temperature for 2 hours. The membrane was then incubated with horseradish-peroxidase-conjugated secondary antibody or Alexa-647-conjugated secondary antibody at 25°C for 1 hour as described previously [68 (link)]. Anti-LANA antibody was purchased from Millipore-Sigma (St. Louis, MO, USA) and an Anti-K-Rta antibody was described previously [69 (link)].