Horseradish peroxidase conjugated anti mouse

Cells were seeded in six-well plates in DMEM. One day later, after washing in DPBS, the cells were treated with diluted sample in DMEM with FBS 2% for pre-determined amounts of time. Following treatment, the cells were washed in DPBS and lysed in RIPA buffer (Noble Bio, Hwaseong, Korea) containing protease inhibitor cocktail (PIC, Sigma Aldrich) and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich) for 30 min at 4°C. The lysate was subjected to centrifugation at 13,000 rpm for 20 min and the resulting supernatant was stored on ice for immediate use or –20°C for longer term storage. The protein content of the supernatant was quantified using a BCA Protein Assay Reagent Kit (Thermo Scientific) with bovine serum albumin as the standard. Equal amounts of protein were separated by NuPAGE™ 12% Bis-Tris Gel (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membranes. Antibodies against β-actin (1:20,000, Sigma Aldrich), p62 (1:1,000, Abcam) were used at 4°C for 24 h. Blots were then incubated with Horse-radish peroxidase conjugated anti-mouse (1:20,000, Bio-Rad, Hercules, CA, USA) or anti-rabbit (1:5,000, Bethyl, Montgomery, TX, USA) secondary antibodies as appropriate at 4°C for 2 h. Blots were visualized by adding a chemiluminescent substrate (Thermo Scientific) and imaged with a FlourChemE imager (HNS Bio, Seoul, Korea).