Western Blotting of p62, Tubulin, and GFP

The polyclonal affinity-purified p62 antibody was raised in rabbits using the peptide antigene PRTEDPVTTPRSTQ corresponding to amino acids 297–311 (Genscript). Polyclonal anti-GFP antibodies were raised using standard procedures by immunizing rats with bacterially expressed His-tagged eGFP purified on Ni affinity columns (Sigma). Samples were separated by SDS-PAGE on an 8% acrylamide gel and transferred to Immobilon-P PVDF membrane (Millipore). Membranes were blocked in 3% milk/TBS for 1 h at room temperature and washed three times for 10 min each in TBST (TBS +0.1% Tween-20). Blots were incubated with primary antibodies: rabbit polyclonal anti-p62 [1∶8,000], mouse monoclonal anti-tubulin AA4.3 (DSHB) [1∶200], rat polyclonal anti-GFP [1∶10,000] in 1.5% milk/TBST for 1 h at room temperature, followed by three 10-min washes in TBST. Blots were incubated in AP-conjugated goat anti-rat (Sigma), anti-rabbit or anti-mouse secondary antibody (Millipore) diluted 1∶10,000 in 1.5% milk/TBST for 1 h at room temperature. Blots were washed for 3×10 min in TBST and then incubated with Immobilon Western Chemiluminescent AP Substrate (Millipore), followed by exposure to Super RX film (Fuji).