Ab124954

Total protein content was extracted from 3 × 105 cells in each group using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid kit (Bioswamp) following the manufacturer's protocol. 20 μg of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies against CDC2 (Bioswamp, PAB30052, 1 : 1,000), B-cell lymphoma-2 (Bcl-2, Bioswamp, PAB30042, 1 : 1,000), Bcl-2-associated X (Bax, Bioswamp, PAB30040, 1 : 1,000), nuclear factor erythroid-2-related factor 2 (Nrf2, Bioswamp, PAB30175, 1 : 1,000), TrxR1 (abcam, ab124954, 1 : 5,000), apoptosis signal regulating kinase 1 (ASK1, Bioswamp, PAB36297-P, 1 : 1,000), phosphorylated (p)-ASK1 (abcam, ab47304, 1 : 1,000), and GAPDH (Bioswamp, PAB36269, 1 : 1,000) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-labeled goat antirabbit IgG secondary antibody (Bioswamp, PAB160011, 1 : 20,000) for 1 h at room temperature and visualized using a Tanon-5200 apparatus (Tanon, Shanghai, China). The band gray values were read using the TANON GIS software (Tanon). GAPDH acted as the internal reference.

The protein levels of TXNRD1 or hypoxia-inducible factor 1α (HIF-1α) and housekeeping Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined using Western blot analysis. In brief, whole cellular protein was extracted from cultured cells by a brief sonication in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany), and fractioned by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for detection of TXNRD1 or 7% SDS-PAGE for HIF-1α. After being transferred to nitrocellulose membranes (Bio-Rad Lab, Hercules, CA, USA), the TXNRD1 proteins (55 kDa) on the blot were identified using rabbit monoclonal anti-TXNRD1 antibody (Ab 124954, Abcam, Toronto, ON, Canada) or HIF-1α (~120 kDa) using mouse monoclonal anti-HIF-1α antibody (sc-53546, Santa Cruz Biotech, Dallas, TX, USA) along with horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were reprobed using housekeeping GAPDH (36 kDa) with anti-GAPDH antibody (Epitope Biotech Inc., Vancouver, BC, Canada) to confirm equal protein loading in each sample. The expression levels of the TXNRD1 or HIF-1α proteins were measured using a densitometry, and were calculated as the ratio of the TXNRD1 or HIF-1α protein to the GAPDH on the same blots.

Skeletal muscle tissues were homogenized in RIPA buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS) with 1% protease inhibitor cocktail (Abcam, ab65621), from which total protein was extracted by centrifugation at 20,000× g. The protein concentration of the extract was measured using a protein assay kit (Pierce; Rockford, IL, USA) and was then adjusted to equal volume in all samples with 2 × 4% SDS sample buffer. The samples were boiled for 5 min and were then loaded on a 7.5% SDS-PAGE gel (30 µg protein/10 µL per well) followed by electrophoresis using a Bio-Rad mini gel apparatus set at 40 mA/gel for 45 min. The fractionated protein on the gel was electrically transferred onto a polyvinyl difluoride membrane (Millipore). The membrane was first probed with the primary antibodies to Sdhd (ab189945) and Txnrd1 (ab124954) purchased from Abcam followed by the secondary antibody (HRP Goat Anti-Rabbit IgG Antibody, HRP, Thermo-Fisher Scientific, Waltham, MA, USA). After three washes with TBST, the membrane was treated with enhanced chemiluminescence substrate (Pierce; Rockford, IL, USA) for 5 min. The blots on the membrane were visualized and analyzed using a UVP BioImaging System (EpiChemi II Darkroom). The final reported data were normalized by Ponceau S staining.