Western Blot Analysis of Cellular Proteins

After lysing the cells in a RIPA buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 1% Triton X-100, 1% NP40, 0.1% SDS, and 0.5% deoxycholate) in the presence of proteinase and phosphatase inhibitors (Bionovas, Toronto, ON, Canada), the proteins in the cell lysates were separated through sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose membranes [35 (link)]. The following antibodies were used: KEAP1 (Abclonal, Woburn, MA, USA), p62 (GeneTex, Hsinchu, Taiwan), phosphorylated (Ser349) p62 (Cell Signaling Technology, Danvers, MA, USA), FLAG tag (Sigma-Aldrich), CBP tag (calmodulin-binding peptide, Santa Cruz, for pNTAP expression vector), ubiquitin (Cell Signaling Technology, Danvers, MA, USA), LSD1 (Cell Signaling Technology, Danvers, MA, USA), Mono-Methyl-Histone H3 (Lys4; H3K4me1, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). The corresponding horseradish peroxidase–conjugated antibodies were obtained from Santa Cruz Biotechnology, and chemiluminescence reagents were acquired from Millipore (Burlington, MA, USA). The signal intensity of autoradiograms was quantified with ImageJ software after normalization to the corresponding GAPDH intensity.