Glut4

In our study, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS solution), 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N,N′,N′-tetramethylethylenediamine (TEMED), acrylamide/bisacrylamide, ammonium persulfate (APS), ascorbic acid (vitamin C), bovine serum albumin (BSA), ethanol (96%), glycine, methanol, Ponceau S, resazurin sodium salt (RES), sodium dodecyl sulfate (SDS), Trolox, trypsin-EDTA solution, Tris-HCl, Tris-Base, and Tween-20 were purchased from Merck KGaA (Darmstadt, Germany). The PVDF membrane with 0.45 µm-pore size and primary antibodies against SOD1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies against GAPDH, NF-κB, IκBα, SRC, GLUT4, PCNA, PGC-1α, PPARγ, p-ERK1/2, and ERK1/2 were purchased from ABClonal (Woburn, MA, USA). The primary antibodies against NLRP3, anti-mouse- and anti-rabbit-HRP-conjugated antibodies were purchased from ThermoFisher (Waltham, MA, USA). Potassium persulfate (Warchem, Zakręt, Poland), antibiotics (Penicillin-Streptomycin, Life Technologies, Bleiswijk, The Netherlands), DMEM (Dulbecco’s Modification of Eagle’s Medium, Biological Industries, Beit Haemek, Israel), FBS (Fetal Bovine Serum, Biological Industries, Genos, Lodz, Poland), and phosphate buffered saline (PBS, pH 7.00 ± 0.05, ChemPur, Piekary Ślaskie, Poland) were also used during the analyses.

First, cells were lyzed on ice in a radioimmunoprecipitation (RIPA) buffer with a protease inhibitor then quantified by quinolinic acid (BCA) assay. Second, SDS-PAGE isolated a total of 50 μg of protein lysate, which was then transferred to a PVDF membrane (Millipore Corp, Billerica, MA, USA). Third, the membrane was blocked with 5% fat-free milk, then incubated with PGK1 (ABclonal Biotech Co., Woburn, MA, USA, 1:100), MYC (ABclonal Biotech Co., Woburn, MA, USA, 1:100), LDHA (ABclonal Biotech Co., Woburn, MA, USA, 1:100), HK2 (ABclonal Biotech Co., Woburn, MA, USA, 1:100), GLUT4 (ABclonal Biotech Co., Woburn, MA, USA, 1:100), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABclonal Biotech Co., Woburn, MA, USA, 1:1000) at 4 °C overnight. After that, the membranes were incubated with secondary antibodies. Then, ECL test reagent (Fudebio, China) was used to show the bands.