Pierce enhanced chemiluminescence reagents

Western blot analyses were performed using standard methods. The following primary antibodies were used: anti-Sox2 (cat. no. 2748; Cell Signaling, Danvers, MA), anti-Notch1 (cat. no. 07-1232; EMD Millipore, Billerica, MA), anti-ID4 (cat. no. sc-13047; Santa Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH (cat. no. MAB374; EMD Millipore, Billerica, MA). Goat anti-rabbit (cat. no. 31462; Thermo Scientific, Pittsburgh, PA) and goat anti-mouse (cat. no. AP124P; EMD Millipore, Billerica, MA) were used as secondary antibodies. For detection, Pierce enhanced chemiluminescence reagents (Thermo Scientific, Pittsburgh, PA) were used according to the supplier’s protocols.

Western blotting was performed as previously described (29 (link)). In brief, after indicated treatments, cells were trypsinized, harvested, and washed with 1X PBS. Pellets were lysed and protein concentrations were determined by the Bradford Assay (Bio-Rad Laboratories, 5000205). Protein samples were loaded and subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and blocked with 5% milk in 1X PBS with 0.1% Tween 20 (Fisher, BP337). The following primary antibodies were used: SQSTM1/p62 (Cell Signaling Technology, 5114T); c-Myc (Cell Signaling Technology, 5605); ATG5 (Cell Signaling Technology, 2630); LC3B (Cell Signaling Technology, 3868); BRD4 (Cell Signaling Technology, 13440S); B-actin (Cell Signaling Technology, 4970); and GAPDH (Cell Signaling Technology, 2118). The membrane was incubated overnight at 4°C with the indicated primary antibodies at a dilution of 1:1000 in 5% BSA. Secondary antibodies: Horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, anti-mouse, 7076S; anti-rabbit, 7074S). The membrane was then washed, secondary antibody was added at a dilution of 1:2000 in 5% BSA for 2 h at room, and the membrane was washed again with 1X PBS with 0.1% Tween 20 three times. Blots were developed using Pierce enhanced chemiluminescence reagents (Thermo Scientific, 32132) on BioRad ChemiDoc System.

Western blotting was performed as previously described [17 (link)]. Briefly, after the indicated treatments, cells were trypsinized, harvested, and washed with 1X PBS. Pellets were lysed and protein concentrations were determined via the Bradford Assay (5000205, Bio-Rad Laboratories; Hercules, CA, USA). Protein samples were loaded and subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and blocked with 5% BSA in 1X PBS with 0.1% Tween 20 (BP337, Fisher; Hampton, NH, USA). The membrane was incubated overnight at 4 °C with the indicated primary antibodies at a dilution of 1:1000 (except for c-Myc 1:250) with 5% BSA in 1X PBS. The membrane was then washed, secondary antibody was added at a dilution of 1:2000 with 5% BSA in 1X PBS for 2 h at room temperature, and the membrane was washed three times in 1X PBS with 0.1% Tween 20. Blots were developed using Pierce enhanced chemiluminescence reagents (32132, Thermo Scientific) on Bio-Rad ChemiDoc System. Image-J software was utilized for quantification of Western blots.