Western blocking buffer

The TGF-β2 cytokine was purchased from Pepro Tech. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH), horseradish peroxidase (HRP)-conjugated secondary antibodies, RIPA buffer, H2DCF-DA, BCA kit, western blocking buffer, H₂DCF-DA were the products of Beyotime (China). The nucleoside 5-aminoimidazole-4-carboxamide riboside (AICAR), metformin hydrochloride, Phorbol 12-myristate 13-acetate (PMA), ML385 (an Nrf2 inhibitor) (CAS No.: 846557-71-9), Atropine (CAS No.: 51-55-8) and Quinidine (CAS No.: 56-54-2) were acquired from MedChemExpress (MCE) (China). Antibodies against AKT, p-AKT, AMPK and p-AMPK (172) were purchased from CST. Antibodies against superoxide dismutase 1 (SOD1), SOD2, γ-glutamyl cysteine synthetase (γ-GCS), Catalase (CAT), smad2/3 and β-actin were purchased from Proteintech. Antibodies against Nrf2, α-smooth muscle actin (α-SMA) (ab5694), Osteopontin (OPN) (ab8448), collagen-I, p-smad2/3 and fibronectin (ab34710) were purchased from Abcam. Antibody against p-Nrf2(S40) was provided by Santa Cruz Biotechnology. The cell culture reagents were the products of Gibco Laboratories (CAS No.:16000-044).

Fetal bovine serum (KGY009), incomplete culture medium (KGM1640SF), and the Nuclei and Cytoplasm Protein Extraction Kit (KGP150) were supplied by KeyGen Biotech (Nanjing, China). The Bradford protein assay kit, Cell Counting Kit-8 (CCK-8 Kit), western blocking buffer, primary antibody dilution buffer, and secondary antibody dilution buffer were from Beyotime Institute of Biotechnology (Haimen, China). Trypsin was from Promega (Madison, USA). Nonlinear immobilized pH gradient (IPG) strips were purchased from GE (Piscataway, USA). Chemicals used for 2DE were purchased from Amresco (Solon, USA). ZnSO₄∙7H₂O was purchased from Aladdin (Shanghai, China). All water used in experiments was Millipore Milli-Q filtered at a resistivity greater than or equal to 18.25 MΩ·cm. Culture dishes, and polyvinylidene difluoride (PVDF) membranes were from Millipore (Bedford, USA). Primary antibodies were purchased from the following vendors: anti-actin antibody (AA128, Beyotime); Hsp90α (D1A7) rabbit mAb (CST#8165) and hnRNPA1 (D21H11) rabbit mAb (CST#8443) (both Cell Signaling Technology). Secondary antibodies, including goat anti-rabbit lgG-HRP (sc-2004) and goat anti-mouse lgG-HRP (sc-2005), were purchased from Santa Cruz Biotechnology.

The total protein of the small intestinal samples was extracted via Protein Extraction Kit (Beyotime, Shanghai, China), then conventional BCA was used to measure total protein concentrations. A PAGE gel was prepared using a PAGE Gel Fast Preparation Kit (EpiZyme, Shanghai, China). After sample addition, electrophoresis was performed using PowerPac Basic (Bio-Rad, Singapore) at 80–110 V. Then, the Immobilon-P transfer membrane (Merck Millipore, Boston, US) was activated in methanol, and proteins were transferred at 200 mA for 2 h. Western Blocking Buffer (Beyotime) was used to block non-specific binding. The membrane was then incubated with primary specific polyclonal rabbit antibodies against ERK1/2, p38, PKR, and Hck (1:2,000; BIOSS, Beijing, China) or β-actin (1:300; Beyotime) at 4°C overnight. After washed using TBS-Tween three times, the membrane was subsequently incubated with secondary antibodies at 4°C for 2 h. A BeyoECL Plus P0081 88 kit were used to color the bands. After captured by FusionCapt Advance FX7 (Fusion FX; OSTC Ltd. San Diego, CA, USA), the images were processed using Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) to acquire quantitative data following the steps published by Wu et al. (28 (link)).

The human glioma samples were obtained from Sun Yat-Sen University Cancer Center, and the relevant analysis of glioma samples were approved by the Ethics Committee of Sun Yat-Sen University Cancer Center. Glioma tissue or cells were lysed using RIPA lysis buffer (Beyotime, China) containing 1% phenylmethanesulfonyl fluoride (PMSF) on ice for 30 minutes and then centrifuged at 12,000 RPM at 4°C for 15 minutes. The supernatants were collected and total protein concentrations determined by using BCA protein assay kit (Pierce, Rockford, IL, USA). We chose 8%, 10% or 12% SDS-gels according to protein molecular weight. After separation by SDS-PAGE, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in western blocking buffer (Beyotime, China) for 2 hours, rinsed with western washing liquid (Beyotime, China) for 1 minute and incubated with the pertinent antibody at 4°C overnight. After washing with western washing liquid 3 times for 10 minutes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. After development and fixing, the protein bands were analyzed by using Image J software, with β-actin or GAPDH as a control.

Cells were lysed over 30 min with RIPA buffer (P0013B, Beyotime, China) containing a protease inhibitor cocktail on ice to harvest total protein. The protein concentration of cell lysate was measured with a BCA protein quantification kit (PC0020, Solarbio, China), according to the manufacturer's protocol. Equal amounts of protein were separated by 5-10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (BD Biosciences, USA). After blocking for 1 h in Western Blocking Buffer (P0023B, Beyotime, China) at room temperature, membranes were incubated with rabbit antibodies: anti-NFATC1 (1:1000, #8032, Cell Signaling Technology, USA), anti-NFATC3 (1:1000, #4998, Cell Signaling Technology, USA), anti-HIF-1α (1:1000, ab179483, Abcam, USA), and anti-GAPDH (1:5000, K106389P, Solarbio, China) at 4 ºC overnight. Membranes were incubated with appropriate horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000, Beyotime, China) for 2 h at room temperature. Finally, signal was detected with SuperSignal West Femto Maximum Sensitivity Substrate (34095, Thermo Scientific, USA) and visualized by VersaDoc (Bio-Rad, USA).

Cell lysis was performed with RIPA buffer (P0013D; Beyotime, Shanghai, China) supplemented with protease (Selleck, Shanghai, China) and phosphatase inhibitors (Selleck). The BCA Protein Assay Kit (PC0020; Solarbio, Beijing, China) was used to measure protein concentrations. The samples were boiled at 100 °C and centrifuged to obtain the supernatant, after which 100 µg of protein was loaded onto 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels (EpiZyme, Shanghai, China). The proteins were transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), and the membranes were blocked in a Western blocking buffer (P0023B; Beyotime) for 2 h at 15 °C. The sections were then incubated with primary antibodies (1:2000) overnight at 4 °C and with the relevant secondary antibody (1:10,000) for 2 h at 15 °C. Bands were visualised using BeyoECL Plus (P0018S; Beyotime). Protein bands were quantified relative to the loading control (GAPDH). The following antibodies were used for Western blotting: anti-GAPDH (AF1186; Beyotime), anti-rabbit-IgG-HRP (A120-111P; Bethyl, Montgomery, TX, USA), and anti-COX2 (ab62331; Abcam, Cambridge, UK).