Polyvinylidene fluoride microporous membrane

Total protein (50 µg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After protein transfer to polyvinylidene fluoride microporous membranes (Bio-Rad), the membranes were blocked with 5% nonfat dry milk and incubated sequentially with the primary antibodies (HIF-1α 1∶500; P-gp 1∶200; β-actin 1∶5000), followed by incubation with the fluorescein-linked anti-mouse (anti-rabbit) IgG (1∶1000) and then incubation with anti-fluorescein alkaline phosphatase-conjugated antibody (1∶5000). All antibodies were bought from Santa Cruz, CA, USA. The immune complexes were detected with the enhanced chemiluminescence reagent (Pierce, USA). For quantification, signals were densitometrically normalized to β-actin by Quantity One image analysis software (Bio-Rad).

The protein was collected from aortas and anti-CD3/CD28 stimulated splenic CD4+ T cells from each group using lysing buffer (PBS with 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/mL phenylmethyl sulfonyl fluoride, 0.3 TIU/mL aprotinin) on ice for 30 mins. Cells or tissue homogenates were then centrifuged at 12,000 g for 15 min at 4°C. Protein concentration was measured in the supernatants using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). 20 μg of protein was separated by SDS-PAGE and transferred into polyvinylidene fluoride microporous membranes (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked with 5% milk in 0.5% Tris-buffer saline solution (pH 7.6) for 1 hour and then incubated with primary antibodies for GARP (Aviva Systems Biology), TGF-β (Santa Cruz's Biotechnology, Inc., for precursor and mature TGF-β1), Smad3/p-Smad3 (Santa Cruz's Biotechnology, Inc.), and GAPDH (EPITOMICS) overnight at 4°C. The membrane was then incubated for 30 mins with HRP conjugated secondary antibody (KPL Co., Ltd.) at room temperature. The blots were visualized using ECL Western Blotting Detection Reagents (Bio-Rad Laboratories, Hercules, CA).

Left ventricular tissues were homogenized at 4° C with a homogenizer in lysis buffer containing 1% Triton X-100, 0.5% deoxycholate, and 5 mmol/L 2-mercaptoethanol. We scraped cell extracts into lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 6 mM urea, and 200 mmol/L potassium chloride with a protease inhibitor cocktail, followed by vigorous vertexing and cooling on ice for 15 min before a 15-min centrifugation at 12,000 g. We then ran the samples on SDS-PAGE. We transferred the proteins on gels onto polyvinylidene fluoride microporous membranes (Bio-Rad, CA, USA) and probed them with primary antibodies against HIF2 α (No. ab109616, Abcam, USA; 1:1000), anti-GAPDH (internal control; No. KC-5G4, Kangcheng, Shanghai, China; 1:8000), anti-IL-6 (No. ab9324, Abcam, USA; 1;1000), anti-AKT (No. #4685, Cell Signaling Technology, USA; 1:2000) and anti-p-AKT (No. #4051, Cell Signaling Technology, USA; 1:2000). The secondary antibodies were coupled with horseradish peroxidase (Cell Signaling Technology, 1:6000). We visualized the immunoreactions using an enhanced ECL detection kit (No. GERPN2209, Amersham Pharmacia Biotech, USA) and then exposed them to film before quantifying them with a video documentation system (Gel Doc 2000, Bio-Rad).

Western blot was performed as described previously [22 (link)]. Left ventricles were homogenized, and the cells were directly lysed in ice-cold RIPA buffer. The samples were analyzed by SDS-PAGE. Transfer the protein to a polyvinylidene fluoride microporous membrane (Bio-Rad) with primary antibodies PFKFB3 (Abcam, USA; 1: 1000), HIF1α (CST, USA; 1: 1000), and anti-GAPDH (internal control; Kangcheng Co., Ltd., China; 1 : 8000). The bands were detected by horseradish peroxidase-coupled secondary antibodies (Cell Signaling Technology, 1 : 6000) and were visualized using the ECL detection kit (Amersham Pharmacia Biotech) and quantified with a video documentation system (Gel Doc 2000, Bio-Rad).

Protein expression was analyzed by Western blot. The total protein (50 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After the protein is transferred to the polyvinylidene fluoride microporous membrane (Bio-Rad), the membrane is blocked with 5% skimmed milk powder and the primary antibody [anti-Bax, anti-PCNA, anti–β-catenin, anti-Drp1 (1:1,000 dilution, American Abcam), anti-NLRP3 (1:500 dilution, American Abcam), anti-ASC, or anti–caspase-1]; then anti-mouse (anti-rabbit (IgG)) is linked with fluorescein (1: 1,000), and then incubated with an antibody conjugated to anti-fluorescein alkaline phosphatase (1:5,000). The immune complexes were detected with enhanced chemiluminescence reagents. For quantification, the signal optical density was normalized to β-actin by Quantity One image analysis software.

Embryos at 72 hpf were homogenized and transferred to RIPA lysis buffer with proteinase inhibitor. Then, the appropriate SDS-PAGE loading buffer was added and the obtained protein was boiled for 10 min. An almost equal amount of protein in each line was separated by polyacrylamide gel electrophoresis. The separated protein was transferred to polyvinylidene fluoride microporous membrane (Bio-Rad Laboratories, Hercules, CA, USA). The blots were blocked with 0.2% skim milk in TBS containing 0.1% Triton X-100, followed by incubation first with the primary antibodies (1:200), and then with secondary antibodies (1:1000). Finally, the blots were visualized using enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA).