Paraffin-embedded blocks of the right middle lobe were sliced into 5 μm thick sections and stained with rabbit anti-rat ACSL4 (1:100, A6826, ABclonal, Woburn, MA, USA). The expression of ACSL4 in lung tissue was detected by HRP rabbit secondary antibody. The sections were deparaffinized with xylene, dehydrated with ethanol, and heated in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was inactivated in 3% H2O2 for 15 min at room temperature. After the sections were incubated in blocking buffer (10% goat serum albumin), they were incubated with primary antibodies recognizing ACSL4 (1:100, A6826, ABclonal, Woburn, MA, USA) at 4 °C overnight. The tissues were then incubated with secondary anti-rabbit antibody-coated polymer peroxidase complexes (A0208, Beyotime, Nanjing, China) at room temperature. After the tissues were incubated with chromogenic substrate DAB (P0203, Beyotime, Nanjing, China) for 1 min, the slides were incubated with hematoxylin (C0107, Beyotime, Nanjing, China) for 10 s. The sections were washed in running water for 20 m. Five fields per slides were randomly chosen by the viewer and semi-quantifiedwith Image j software.
A6826
Manufactured by ABclonal
Sourced in China, United States
A6826 is a laboratory equipment product. It serves as a device for performing specific tasks related to scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
A1933, by ABclonal (3 mentions)
A2413, by ABclonal (2 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
C0107, by Beyotime (1 mentions)
A0208, by Beyotime (1 mentions)
Molecular imager gel doc xr system, by Bio-Rad (1 mentions)
R0010, by Solarbio (1 mentions)
P1040, by Solarbio (1 mentions)
Hvlp04700, by Merck Group (1 mentions)
A5865, by ABclonal (1 mentions)
As014, by ABclonal (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
A3854, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab204916, by Abcam (1 mentions)
Tanon 5200 multiimage system, by Shanghai Tanon Science & Technology (1 mentions)
Wla019, by Wanlei (1 mentions)
A11243, by ABclonal (1 mentions)
8 protocols using a6826
1
Immunohistochemical Analysis of ACSL4 in Lung Tissue
2
Protein Extraction and Western Blot Analysis
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Proteins were extracted from freshly frozen tissues. Briefly, the tissues were homogenized and lysed with radio immunoprecipitation lysis buffer (Solarbio, R0010, Beijing, China), which has 100 mg/ml of phenylmethanesulfonyl fluoride (ADOOQ, A11901, California, USA) and 1 mg/ml of Aprotinin (ADOOQ, A14263). The lysate was collected and centrifugated for liquid supernatant. A total of 50 μg protein samples were separated with 10% SDS-PAGE (Solarbio, P1040) and transferred onto polyvinylidene fluoride (Millipore, HVLP04700, Arklow, Ireland) membranes. Membranes were blocked at room temperature with 5% skim milk (ACMEC, AC11037, Shanghai, China) powder in TBS (Quartett, 402000192, Berlin, Germany) for 1 h. Membranes were then incubated overnight at 4 °C with primary antibodies against the following proteins: Acyl-CoA synthetase long-chain familymember 4 (ACSL4) (1: 1000, ABclonal, A6826, Wuhan, China), transferrin receptor (TFRC) (1: 1000, ABclonal, A5865, Wuhan, China). Secondary antibodies (1: 1000, ABclonal, AS014, Wuhan, China) were incubated for 1 h at 37 °C and washed three times with PBST (PBS+0.1% Tween 20) (Biomed, PA202, Beijing, China). The protein bands were visualized by Molecular Imager Gel Doc XR System (Bio-Rad, Hertfordshire, UK) and quantified by densitometric analysis using an image analyzer (NIH Image J software, Bethesda, USA).
3
Western Blot Analysis of Ferroptosis Markers
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The cells were cleaned with 1 × PBS before being lysed with M-PER mammalian protein extraction reagent (78501, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with a Cocktail (11873580001, Roche, Basle, Switzerland). Protein concentrations were measured as indicated by the manufacturer using the BCA protein assay kit (23250, Thermo Fisher Scientific, Waltham, MA, USA).
Protein samples (30 μg) were separated in SDS-PAGE gels ranging from 4% to 15% and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, San Francisco, CA, USA). After blocking in 5% skim milk for 60 min, the membranes were treated with the appropriate primary antibodies and fluorescent-conjugated secondary antibodies. Primary antibodies against SRSF2 (ab204916, 1:1000, Abcam, Cambridge, UK), SLC7A11 (A2413, 1:1000, ABclonal, Wuhan, China), ACSL4 (A6826, 1:1000, ABclonal, Wuhan, China), and GPX4 (A1933, 1:1000, ABclonal, Wuhan, China) were used. The internal standard was probed with an anti-β-actin-peroxidase monoclonal antibody (A3854, Sigma-Aldrich, Bedford, MA, USA). In a Tanon 5200 MultiImage System (Tanon Science & Technology, Shanghai, China), ECL reagents (34080, Millipore, Burlington, MA, USA) were used to observe the bands. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to calculate band intensities.
Protein samples (30 μg) were separated in SDS-PAGE gels ranging from 4% to 15% and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, San Francisco, CA, USA). After blocking in 5% skim milk for 60 min, the membranes were treated with the appropriate primary antibodies and fluorescent-conjugated secondary antibodies. Primary antibodies against SRSF2 (ab204916, 1:1000, Abcam, Cambridge, UK), SLC7A11 (A2413, 1:1000, ABclonal, Wuhan, China), ACSL4 (A6826, 1:1000, ABclonal, Wuhan, China), and GPX4 (A1933, 1:1000, ABclonal, Wuhan, China) were used. The internal standard was probed with an anti-β-actin-peroxidase monoclonal antibody (A3854, Sigma-Aldrich, Bedford, MA, USA). In a Tanon 5200 MultiImage System (Tanon Science & Technology, Shanghai, China), ECL reagents (34080, Millipore, Burlington, MA, USA) were used to observe the bands. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to calculate band intensities.
4
Quantitative Western Blot Analysis
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Total proteins were extracted from tissues or cells using a lysis buffer (Wanleibio, WLA019, Shenyang, China). Proteins were quantified using a BCA protein assay kit (Wanleibio, WLA004, Shenyang, China). The denatured proteins are then separated by SDS-PAGE followed by an electrophoretic transfer onto a PVDF membrane. After blocking with 5% nonfat dry milk at room temperature for an hour, the membranes were incubated with the primary antibodies against ACSL4 (Abclonal, A6826, Wuhan, Hubei, China), glutathione peroxidase 4 (GPX4) (Abclonal, A11243, Wuhan, Hubei, China) and ferritin heavy chain (FTH) (Wanleibio, WL05360, Shenyang, China) at 4 °C. The membranes were then incubated with diluted secondary antibodies (1: 5000, Wanleibio, WLA023, Shenyang, China). Quantification of band intensities was performed using Gel-Pro-Analyser software.
5
Western Blot Analysis of Lipid Metabolism Proteins
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The protein extraction and western blot were described in our previous study [30 (link)]. The specific primary antibodies are as follows: rabbit anti-ACSL4 (A6826, 1 : 1000, ABclonal, Wuhan, China), rabbit anti-LPCAT3 (A17604, 1 : 1000, ABclonal, Wuhan, China), rabbit anti-LOXs (A11504, 1 : 1000, ABclonal, Wuhan, China), rabbit anti-POR (YN1836, 1 : 1000, Immunoway, Plano, TX, USA), and mouse anti-GAPDH (RM2002, 1 : 10000, Beijing Ray Antibody Biotech, Beijing, China). The protein signals were detected after using the chemiluminescent substrate kit (Millipore, Darmstadt, Germany) and then analyzed by ImageJ software (NIH, Bethesda, MD, USA). Each experiment was repeated three times, and all protein expression was normalized to that of GAPDH.
6
Comprehensive Western Blot Analysis of EMT and Ferroptosis Markers
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The antibodies included the following: anti-E-cadherin (20874-1-AP, Proteintech, Wuhan), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan), anti-SNAI1 (13099-1-AP, Proteintech, Wuhan), anti-Vimentin (10366-1-AP, Proteintech, Wuhan), anti-GPX4 (A1933, Abclonal, Wuhan), anti-β-tubulin (M20005, Abmart, Shanghai), anti-AKT (60203-2-Ig, Proteintech, Wuhan), anti- phospho-AKT (Thr308) (29163-1-AP, Proteintech, Wuhan), anti-mTOR (66888-1-Ig, Proteintech, Wuhan), anti-phospho-mTOR (67778-1-Ig, Proteintech, Wuhan), anti- eIF4EBP1 (A19045, Abclonal, Wuhan), anti-phospho-eIF4EBP1 (AP0030, Abclonal, Wuhan), anti-Ki67 (GB121141, Servicebio, Wuhan), anti-SLC7A11 (26864-1-AP, Proteintech, Wuhan), anti-ACSL4 (A6826, Abclonal, Wuhan), and anti-FTL (ab69090, Abcam, UK). The AKT activator SC79 (HY-18749) and mTOR activator MHY1485 (HY-B0795) were purchased from MCE (USA). P28 peptide (sequence: LSTAADMQGVVTDGMASGLDKDYLKPDDC) was purchased from GenScript. Chloroquine (CQ), deferoxamine (DFO), and the proteasome inhibitor MG132 were purchased from MCE. The apoptosis inhibitor Z-VAD-FMK (S7023), the ferroptosis inhibitor ferrostatin-1 (S7243), and the necrosis inhibitor necrosulfonamide (S8251) were purchased from Selleck (USA). Cycloheximide was obtained from Sigma‐Aldrich.
7
Dapagliflozin and Ferroptosis Inhibition
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Dapagliflozin was obtained from MCE (Cat. HY-10450). Ferrostatin-1 (Fer-1) was purchased from Selleck Chemicals (Cat# S7243). Drugs were separately dissolved in DMSO and added to the culture medium 12 h before H/R. For in vivo experiments, DAPA was purchased from commercially available dapagliflozin tablets (Forxiga Tab 10 mg, AstraZeneca AB, United States). DAPA was diluted with sterile water for injection (WFI) and given to animals by intragastric administration, as previously shown (Nikolaou et al., 2022 (link)). Primary antibodies against β-tubulin (1:5,000, Cat. AC021), GPX4 (1:1,000, A1933), SLC7A11 (1:1,000,A2413), ACSL4 (1:1,000, A6826), PTGS2 (1:1,000, A1253), and FTH1 (1:1,000, A19544) were purchased from ABclonal (Wuhan, China); FTMT (1:200,aa65-227) was purchased from LifeSpan BioSciences (Seattle, WA, United States); p-ERK (1:1,000,#4370), ERK (1:1,000, #4695), p-P38 (1:1,000, #4511), and P38 (1:1,000, #8690) were purchased from Cell Signaling Technology (Beverly, MA, United States); p-JNK (1:1,000, ab76572)), and JNK (1:1,000, ab208035) were purchased from Abcam (San Francisco, CA).
8
Quantification of Kidney Oxidative Stress Markers
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Mouse kidneys were homogenized and centrifuged at 13000 rpm at 4°C for 30 minutes to take the supernatant. Extracts of the kidney tissue were prepared in lysis buffer. The total protein was separated on 5-20% acrylamide gel by using twelve alkyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinyl fluoride two (PVDF). After sealing with 5% fat-free milk for 1 hour, the membrane was incubated with the corresponding primary anti-rabbit antibody at 4°C overnight. Whole-cell lysates of the kidney were analyzed using western blotting to detect the expression of NOX2, NOX4, ACSL4, GPX4, FTH1, and β-actin. Primary antibodies against Gp91phox/NOX2 (1 : 1000; ab129068, Abcam Technology, United Kingdom), NOX4 (1 : 1000; ab133303, Abcam Technology, United Kingdom), ACSL4 (1 : 1000; A6826, ABclonal Technology, China), GPX4 (1 : 1500; ab125066, Abcam Technology, United Kingdom), FTH1 (1 : 1000; ab75972, Abcam Technology, United Kingdom), and β-actin (1 : 10000; AC026, ABclonal Technology, China) were used. The imprinting was visualized by enhanced chemiluminescence (ECL) system, and the gray value was scanned and quantified by ImageJ software.
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