Western Blotting Protein Expression Analysis

Western blotting was performed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system as previously described [9 (link), 10 (link)]. Immunoblotting was performed following incubation of samples with CUL4A (clone EPR3198, 1:10000, Abcam, Cambridge, MA), LC3B (catalog no. NB100–2220, polyclonal, 1:2000, Novus Biologicals, Littleton, CO), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; clone 6C5, 1:10000, Millipore, Burlington, MA) antibodies at 25°C for 2 h. The blots were washed and incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson, West Grove, PA, United States), followed by three washes with TBST (Tris-buffered saline + Tween). Enhanced chemiluminescent HRP substrate (Pierce, Rockford, IL, United States) was used for detection according to manufacturer’s description.