Sc 2054

Western blot analysis was performed as previously described else where (Wang et al., 2017b (link), 2018 (link)). Granulosa cells were collected after treatment for 48 h and lysed in RIPA buffer (catalog number: 89900; ThermoFisher, Rochford, IL, USA), then denatured by boiling for 5 min with bromophenol blue and frozen at −80 °C. The proteins were separated by 12% polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Firstly, the membrane were incubated with primary antibody: Bcl2 mouse monoclonal antibody (1:500, sc-7382; Santa Cruz, Dallas, TX, USA), Bax mouse monoclonal antibody (1:500, sc-20067; Santa Cruz, Dallas, TX, USA), caspase-3 rabbit polyclonal antibody (ab13847; Abcam, California, USA) and β-actin mouse monoclonal antibody (1:1000, SC-47778; Santa Cruz, Dallas, TX, USA). Later, the membrane was detected by incubation with HRP labeled goat anti-rabbit secondary antibody (SC-2054; Santa Cruz, Dallas, TX, USA) or goat anti-mouse secondary antibody (1:5000; SC-2005; Santa Cruz, Dallas, TX, USA), respectively. Finally, membranes was incubated with the Clarity Western ECL kit (catalog number: 170-5060; Bio-Rad Laboratories, Hercules, CA, USA), and scanned in a ChemiDocXRS chemiluminescent imaging system (Bio-Rad, Hercules, CA, USA).

Western blotting was performed using standard procedures. Briefly, samples were loaded into wells in Tris-HCl gels and run at 60V for 10 min initially and then switched to 120V. Protein in the gel was then transferred onto nitrocellulose at 90 V for 2 hr. Membranes were then blocked for 1 hr in 5% non-fat dry milk (NFDM) in TBST, probed overnight at 4°C with primary antibody, rinsed 4 x 5 min in TBST, probed with appropriate secondary antibody for 1 hr at room temperature, and then rinsed in TBST 4 x 5 min. Primary antibodies included HIF1α (Novus Biologics, NB100–105), LDH-A (Santa Cruz sc-27230), IDH (Abcam, ab36329), phosphoglycerate mutase 1 (PGAM1) (Cell Signaling 7534S), cyclin D1 (Cell Signaling 2922), phospho Rb S780 (Cell Signaling 9307S), phospho acetyl CoA carboxylase (ACC) (Millipore 07–303), and ACC (Jackson 016–050–084). The secondary antibodies used with appropriate primary antibody included anti rabbit (Santa Cruz, sc-2054), anti mouse (Santa Cruz, sc-2055), or anti goat (Santa Cruz, sc-2056). Chemiluminescent detection was then performed using enhanced chemiluminescence (ECL) and was detected using autoradiography film. Western blots were analyzed using Image J. Ponceau red staining was used to correct for any variation in protein loading between samples. Values presented in graphs were normalized against the BSA group.

The whole cell lysates were prepared by using RIPA Lysis and Extraction Buffer (Thermo Scientific, IL, USA) with Pierce Protease and Phosphatase Inhibitor Mini (Thermo Scientific, IL, USA). The fractionated lysates were prepared by using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, IL, USA). Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad; CA, USA), and the concentration in individual samples was equalized before adding 4x Laemmli buffer to a final concentration of 1x. Equal amounts of protein (50 μg whole cell lysates, 45 μg fractionated lysates) were run on 10% SDS-PAGE gels and then transferred with iBlot 2 Transfer Stacks and iBlot 2 Dry Blotting System onto PVDF membranes (Invitrogen, Thermo Scientific, IL, USA). After one hour 5% milk block, membranes were probed with the antibodies diluted with 5% BSA (NRF3) or 5% milk (β-actin and PCNA and secondary antibodies) in Tris-buffered saline with 0.1% Tween 20. Primary antibodies anti-NRF3 (HPA055889, Sigma-Aldrich), anti-β-actin (NB600-501, Novus Biologicals, UK), and anti-PCNA (NB600-501SS, Novus Biologicals, UK) were incubated overnight, and appropriate HRP-conjugated secondary antibodies (sc-2054 and sc-2055, Santa Cruz, CA, USA) were incubated at RT for one hour. Blots were detected with the Western blot imaging system Azure 600 (Azure Biosystems, CA, USA).