Anti-HA monoclonal antibody was purchased from MBL (Tokyo, Japan), and anti-Hrd1 and anti-Cdc48 polyclonal antibodies were described previously (Nakatsukasa et al. 2013 (link)). Anti-Pgk1 monoclonal antibody was purchased from Invitrogen.
Proteins were transferred from polyacrylamide gels to Immobilon-P (Millipore) using GENIE Electrophoretic Transfer device (Idea Scientific Company) in blotting buffer (25 mM Tris, 192 mM glycine, 10% methanol) at a constant current of 650 mA. The membranes were washed with TBS-T buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and blocked with 3% skim milk in TBS-T buffer for 30 min. The membranes were incubated with primary antibodies in TBS-T buffer overnight at 4 °C and washed three times with TBS-T (10–60 min for each wash). The membranes were then incubated with secondary antibodies for ~60 min and washed three times with TBS-T. The membranes were incubated with Chemi-Lumi One (nacalai tesque) or Luminata Forte Western HRP substrate (Millipore) and exposed to X-ray film. The band intensities were quantified with ImageJ (NIH). Immunoblots were decorated with the indicated primary antibodies and appropriate HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Sigma Aldrich).
Proteins were transferred from polyacrylamide gels to Immobilon-P (Millipore) using GENIE Electrophoretic Transfer device (Idea Scientific Company) in blotting buffer (25 mM Tris, 192 mM glycine, 10% methanol) at a constant current of 650 mA. The membranes were washed with TBS-T buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and blocked with 3% skim milk in TBS-T buffer for 30 min. The membranes were incubated with primary antibodies in TBS-T buffer overnight at 4 °C and washed three times with TBS-T (10–60 min for each wash). The membranes were then incubated with secondary antibodies for ~60 min and washed three times with TBS-T. The membranes were incubated with Chemi-Lumi One (nacalai tesque) or Luminata Forte Western HRP substrate (Millipore) and exposed to X-ray film. The band intensities were quantified with ImageJ (NIH). Immunoblots were decorated with the indicated primary antibodies and appropriate HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Sigma Aldrich).