Western blot analysis of LC3 and p62 in ovarian cancer cells

The ovarian cancer cells were seeded in a 6-well plate and cultured for 24 h in complete medium with 10% FBS as a negative control for autophagy, or 1% FBS containing medium as a positive control for autophagy, or in OVCAF-CM or OVNF-CM, as indicated. Chloroquine (CLQ; 30 µM) (Sigma-Aldrich; Merck KGaA) was added to CM 8 h prior to cell collection. The cells were harvested and washed with 1X PBS, and lysed with in-house made radioimmuno-precipitation assay (RIPA) buffer containing 150 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0 and then heated for 5 min at 95°C to denature proteins. Protein samples were separated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes by TE 70 Semi-Dry transfer units (GE Healthcare) for 1.5 h. The membranes were blocked in a blocking solution containing 5% (w/v) skim milk in 0.1% (v/v) Tween 20 in TBS-T for 1 h at room temperature. The blots were probed by overnight incubation at 4°C with rabbit anti-human LC3B antibody (1:1,000, L7543, Sigma-Aldrich; Merck KGaA) or rabbit anti-human p62 antibody (1:500, ab109012, Abcam). The blots were then washed in TBS-T before being incubated with 1:2,000 goat anti-rabbit IgG-HRP (ab6721, Abcam) for 1 h. For β-actin detection, mouse anti-human β-actin monoclonal antibody (1:5,000) (sc-47778, Santa Cruz Biotechnology Inc.) and 1:2,000 HRP-conjugated horse anti-mouse IgG antibody (7076, Cell Signaling Technology Inc.) in blocking solution were used as primary and secondary antibody, respectively. The protein bands were developed by adding Clarity™ Western ECL substrate (170-5061, Bio-Rad Laboratories, Inc.) and detected under Gel Document (Syngene^®). The band intensities were measured using ImageJ software version 1.52a. The level of β-actin was used as an internal control to ensure equal amounts of loading proteins.