Ab80588

Western blots were performed as previously described [25 (link)]. Total protein was extracted from kidney tissue in RIPA lysis buffer (25 mM Tris-HCl, 25 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, and 0.1% SDS) with 1% PMSF protease inhibitors (P1005, Beyotime Biotechnology, China) and phosphatase inhibitors (P1081, Beyotime Biotechnology, China) added. Equal amounts of protein were separated by 10-15% SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore, Germany). After blocking with 5% nonfat milk for 3 h, the membranes were incubated with the primary antibodies including TGF-β1, α-SMA, Nrf2, HO-1, NQO1, ASC, caspase-1 (ab215715, ab32575, ab137550, ab13248, ab80588, ab175449, ab1872, Abcam, UK), p-Smad2, Smad2, p-Smad3, Smad3, E-cadherin, NLRP3 ((#18338, #5339, #9520, #9523, #3195, #15101, Cell Signaling Technology, USA), and cleaved caspase-1 (AF4005, Affinity Biosciences, USA) at 4°C overnight. Then, the membranes were washed 3 times with TBST and incubated with the secondary antibodies for 1 h at room temperature. After washing with TBST for a further three times, the protein bands were visualized by enhanced chemiluminescence (32134, Thermo, USA) solution and imaged with Automated Imaging System (Gene Gnome5, Synoptics Ltd, UK). GAPDH or β-actin was assumed to be similarly abundant in all samples and was used as a loading control.

Total protein and nucleoprotein (for Nrf2 and β-TrCP) in the ipsilateral peri-infarct hemisphere were extracted, and the protein concentration was determined by bicinchoninic acid protein (BCA) assay. The protein samples were then denatured by boiling for 10 min. Then, 20 μg of each protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V for 100 min. After transfer to polyvinylidene fluoride (PVDF) membrane, the separated protein bands were incubated with individual primary antibodies (caspase-3 1:500, ab197202, Abcam; AKT, 1:1000, Cat# 9272, CST; p-AKT, 1:1000, Cat#9271, CST; GSK-3β, 1:1000, Cat# 9315, CST; p-GSK-3β, 1:1000, Cat# 9336, CST; β-TrCP, 1:1000, Cat# 4394, CST; Nrf2 1:1000, ab92946, Abcam; PCNA, 1:1000, ab92552, Abcam; HO-1, 1:2000, ab52947, Abcam; NQO1, 1:10000, ab80588, Abcam) at 4 °C overnight, followed by incubation with corresponding secondary antibodies at room temperature for 2 h. The developed bands were observed under a gel imaging system.

METH (purity ≥ 99.1%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ICS (Figure 1(a)) was purchased from the ZZBIO Co., Ltd. (Shanghai, China), and ML385 was purchased from the Macklin Biochemical Co., Ltd. (Shanghai, China). The antibodies used in this study were TH (AB152, Millipore, Germany), DAT (bs-1714R, Bioss, China), α-syn (A7215, Abclonal, China), GFAP (16825-1-AP, Proteintech, China), Iba1 (ab220815, Abcam, USA), Keap1 (60027-1-Ig, Proteintech, China), Nrf2 (66504-1-Ig, Proteintech, China), HO-1 (70081S, CST, USA), NQO1 (ab80588, Abcam, USA), β-actin (bs-0061R, Bioss, China), and corresponding secondary antibodies (BL003A and BL001A, Biosharp, China).

The cytoplasmic and nuclear protein samples were resolved in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto nitrocellulose membrane (IPFL00010; Millipore). The membrane was incubated with primary antibodies at a dilution of 1 : 100 to 1 : 1000 at 4°C for 12 hours, washed out with TBS/T (Tris-buffered saline containing 0.2% Tween 20), exposed to HRP-conjugated anti-goat or anti-mouse secondary antibody (1 : 5000) for 1 hour, respectively, and then visualized by enhanced chemiluminescence detection reagents. Relative intensities of protein bands were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). Antibodies used in this study were as follows: anti-Nrf2 (Ab62352; Abcam), anti-Histone H3 (PAB33309; BIOSWAMP), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, FNab03345; Wuhan Fine Biotech Co., Ltd.), anti-NAD(P)H: quinone oxidoreductase 1 (NQO1) (Ab80588; Abcam), anti-Bax (FNab00810; Wuhan Fine Biotech Co., Ltd), anti-Bcl-2 (658701; BioLegend), anti-Keap1 (Ab139729; Abcam), anti-p-p38 (4511 T; Cell Signaling Technology), anti-p38 (622403; BioLegend), and anti-beta-actin (PAB36265; BIOSWAMP).

Proteins were extracted from cardiac tissue with radio immunoprecipitation assay (RIPA) lysis buffer (P0013 B, Beyotime) and their concentrations were determined with a bicinchoninic acid (BCA) protein kit (P0010S, Beyotime). The protein samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% skim milk powder in TBS + tween (TBST) for 1 h and probed with primary antibodies (GAPDH, ab181602, Abcam; MMP-2, ab37150, Abcam; VEGFA, ab46154, Abcam; Nrf2, ab92946, Abcam; HO-1, ab13243, Abcam; NQO1, ab80588, Abcam) overnight at 4°C and shaken and rinsed three times with TBST solution. Then, the blot was incubated with diluted secondary antibodies (horseradish enzyme-labeled goat antirabbit IgG (H + L), ZB-2301, Chinese fir golden bridge) for 2 h. After washing, enhanced chemiluminescence (ECL) luminescence agent (P0018AS, Biyuntian) was evenly instilled. Finally, the FliorchemHD2 imaging system was used for scanning, analyzing, and collecting images as well as measuring the gray values of each band through Image J.

The cells were lysed on ice in buffer containing protease inhibitors (AS1008, Aspen). The protein fractions were collected and separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). Then, the proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). The membranes were blocked with 5% skim milk. Then, primary antibodies were added and incubated overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The antibodies used in this study included anti-Nrf2 (1:1000, PA5-27882, Thermo Fisher Scientific), anti-β-Actin (1:10,000, TDY051, TDY Biotech), anti-Histone H3 (1:10,000, #4499, Cell Signaling Technology), anti-Drp1 (1:1000, #8570, Cell Signaling Technology), anti-p-Drp1 (Ser616) (1:500, PA5-106169, Thermo Fisher Scientific), anti-VDAC1 (1:3000, ab15895, Abcam), anti-NOQ1 (1:3000, ab80588, Abcam), anti-HO-1 (1:5000, 10701-1-AP, Proteintech Group), anti-SOD2 (1:3000, ab68155, Abcam), anti-cleaved Caspase-3 (1:1000, AF7022, Affinity Biosciences), anti-Bax (1:2000, #2772, Cell Signaling Technology) and anti-Bcl-2 (1:1000, ab19649, Abcam).