The kidney sections were ground using a homogenizer or the cells were scratched and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with proteinase inhibitor (Roche, Switzerland) for 30 min on ice. Protein concentration was quantified using the Bicinchoninic acid assay (Beyotime, Shanghai, China). Protein samples (50 μg) were used to perform immunoblotting as described previously42 (link). The specific primary antibodies used were: anti-GSDME (ab215191, Abcam, 1:1000), anti-caspase-3 (9662, CST, 1:1000), anti-cleaved caspase-3 (9661, CST, 1:1000), phospho-NF-κB p65 (3033, CST, 1:1000), NGAL (ab63929, Abcam, 1:1000), KIM-1 (AF1817, R&D systems, 1:1000), and anti-GAPDH (5174, CST, 1:10000). Peroxidase-conjugated goat anti-rabbit secondary antibodies (A0277, Beyotime, Shanghai, China) were used for immunoblotting.
Ab215191
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab215191 is a laboratory equipment product offered by Abcam. It serves as a core function for laboratory applications. No further details are available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab209845, by Abcam (7 mentions)
Ab219800, by Abcam (5 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ab179515, by Abcam (4 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
West femto substrate, by Thermo Fisher Scientific (2 mentions)
Ab18256, by Abcam (2 mentions)
Anti caspase 3 9662s, by Cell Signaling Technology (2 mentions)
Anti β actin, by Developmental Studies Hybridoma Bank (2 mentions)
T5168, by Merck Group (2 mentions)
F1804, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Ab181602, by Abcam (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ag 20b 0010, by Adipogen (2 mentions)
Ab196436, by Abcam (2 mentions)
37 protocols using ab215191
1
Immunoblotting for Kidney Injury Markers
2
Flow Cytometry Analysis of ADC and Mesothelin
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EMT6 and CT26 cell lines were stained for surface ADC binding using 1 µg/mL of mesoADC or isotype ADC or surface mesothelin expression with 10 µg/mL antimesothelin antibody (AM26533AF-N, Origene) or rat IgGa isotype control (559073, BD Pharmingen) followed by 2 µg/mL goat antimouse IgG Alexa Fluor 647 (421236, Life Technologies) or 4 µg/mL goat antirat IgG Alexa Fluor 488 (ab150165, Abcam). To detect intracellular GSDME expression, cells were fixed in 10% neutral buffered formalin (Sigma) then permeabilized with ice-cold methanol. Cells were stained with 1 µg/mL anti-GSDME antibody (ab215191, Abcam) or rabbit IgG isotype control (02–6102, Invitrogen) followed by 2 µg/mL goat antirabbit Alexa Fluor 647 (A32733, Life Technologies). Data acquisition was performed using a BD Accuri C6 Plus flow cytometer (BD Biosciences), and data analysis was performed using FCS Express software (De Novo Software).
3
Western Blot Analysis of Cell Lysates
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Cells were lysed in lysis buffer (50 mM Tris-Cl pH7.4, 150 mM NaCl, 1% Triton-X100 supplemented with 1 mM PMSF before use). Cell lysate in SDS loading buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% Bromophenol blue) was boiled and analyzed by SDS-PAGE (Bio-Rad) and transfer to PVDF membranes (MilliporeSigma), which were then blocked with 2% milk in TBST buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.6, 0.1% Tween-20) for 30 min before incubation with primary antibodies for 2 hr at room temperature or 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibody for 40 min and then washed 3 times with TBST before adding the West Femto substrate (ThermoFisher Scientific) for detection. Antibodies were anti-GSDME (ab215191, Abcam), anti-FLAG (F1804, Sigma), anti-caspase-3 (9662S, Cell Signaling), anti-HMGB1 (ab18256, Abcam), anti-α-tubulin (T5168, Sigma), and anti-β-actin (JLA20, Developmental Studies Hybridoma Bank).
4
Western Blot Analysis of Apoptosis Regulators
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The total proteins were lysed using a lysate buffer (Beyotime, Shanghai, China) for 30 minutes and washed with PBS. Polyacrylamide gel electrophoresis was employed to separate the protein, which was then loaded onto a polyvinylidene fluoride membrane (Millipore). Non-specific antigens were blocked with 5% skim milk for 2 h, followed by immunoblotting with different antibodies, including GSDME (1:1000, ab215191, Abcam), cleaved Caspase-3 (1:1000, #9664, CST), Glutathione Peroxidase 4 (1:1000, ab125066, Abcam), Transferrin Receptor (1:1000, ab214039, Abcam), xCT (1:1000, T57046, Abmart), GAPDH (1:5000, 60004-1-Ig, Proteintech).
5
Antibody-Based Apoptosis Signaling Assay
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Antibodies for GSDME (ab215191), GSDMD (ab209845) were purchased from Abcam (Cambridge, Cambridgeshire, Britain). Antibodies for caspase-1 (3866T), caspase-9 (9502T), caspase-3 (9668T), caspase-8 (9746T) and γH2AX (9718T) were purchased from Cell Signaling Technology (Danver, MA, USA), and GAPDH (60004-1-lg) was from Proteintech Group (Wuhan, Hubei, China). The secondary antibodies including HRP Goat Anti-Mouse IgG (H+L) (AS003), HRP Goat Anti-Rabbit IgG (H+L) (AS014) and FITC Goat Anti-Rabbit IgG (H+L) (AS011) were bought from Abclonal (Wuhan, Hubei, China).
Other reagents were purchased as follows: caspase-3 inhibitor (Z-DEVD-FMK) (HY-12466) and ROS scavenger N-acetyl cysteine (NAC) (HY-B0215) were from MedChemExpress (Monmouth Junction, NJ, USA); protease inhibitor cocktail tablets (BL612A) were from Biosharp (Beijing, China).
Other reagents were purchased as follows: caspase-3 inhibitor (Z-DEVD-FMK) (HY-12466) and ROS scavenger N-acetyl cysteine (NAC) (HY-B0215) were from MedChemExpress (Monmouth Junction, NJ, USA); protease inhibitor cocktail tablets (BL612A) were from Biosharp (Beijing, China).
6
Immunohistochemical Analysis of GSDME in Renal Biopsies
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Fixed, paraffin-embedded human renal biopsy specimens (1.5-μm thick) were deparaffinized, rehydrated, and blocked with 1.5% H2O2–methanol. After washing with phosphate-buffered saline (PBS), the slides were subjected to antigen retrieval in citrate buffer. Non-specific binding was blocked with 10% donkey serum for 30 min. After that, the slides were incubated with a rabbit anti-GSDME antibody (1:100, Cat# ab215191, Abcam, MA, USA) overnight at 4 °C. Then, the donkey anti-rabbit/mouse antibody was incubated for 30 min and washed with PBS. After staining with 3,3′-diaminobenzidine (DAB), the slides were counterstained with hematoxylin and examined under a microscope (Leica DMLB, Wetzlar, Germany).
7
Protein Extraction and Analysis Protocol
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We lysed cells with RIPA buffer (beyotime, China) and extracted proteins from cells. Then, BCA assay kit (Invitrogen) measured the protein concentration. 10% SDS-PAGE was employed to separate proteins. The protein was transferred to the PVDF membrane (Bio-Rad, USA), the PVDF membrane was blocked with 5% milk, and then incubated with a primary antibody. And then incubated with an NLRP3 (Abcam; ab263899; dilution 1:1000); GSDME (Abcam; ab215191; dilution 1:1000); or GAPDH antibody (Abcam; ab181602; dilution 1:10,000) at 4 °C overnight. After washing the membrane, it was incubated with goat anti-rabbit IgG-HRP for 1–2 h. The photos were taken using Odyssey infrared Fluorescence Western blotting system (Li-Cor Biosciences), and the quantitative pictures was obtained via ImageJ software.
8
Quantifying GSDME and Hsp90 in Lenvatinib-Treated Huh7 Cells
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The total proteins in Huh7 cells treated with Lenvatinib were extracted by RIPA buffer containing 1% PMSF (Beyotime) and quantified by the BCA method (Beyotime). Twenty microgram proteins were used for western blotting against GSDME (1:2000, ab215191, Abcam) and Hsp90 (1:2000, 610419, BD Bioscience).
9
Immunoblotting Analysis of Apoptosis Signaling
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Cells were lysed by radio immunoprecipitation assay lysis buffer and proteins (30 μg) were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). After blocked with blocking buffer (Beyotime) at room temperature for 30 min, the membranes were incubated with primary antibodies against p-Akt (1:1000; #4060, cst), Akt1 (1:1000; #2938, cst), NF-κB p65 (1:1000; #8242, cst), NF-κB p-p65 (1:1000; #ab86299, Abcam), cleaved caspase-3 (1:1000; #9664, cst), caspase-3 (1:1000; #9662, cst), GSDME (1:1000; ab215191, Abcam) and GAPDH (1:1000; #ab181602, Abcam) at 4 °C overnight. After incubation, the membranes were washed and incubated with HRP-conjugated secondary antibodies at room temperature for 2 h and visualized using the ECL system (Beyotime). Protein levels were normalized to GAPDH.
10
Caspase-3 and GSDME Detection
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Cells were collected and sonicated by Sonicator (Diagenode, Bioruptor plus) in 1 × SDS loading (2% SDS, 25 mM Tris base/pH 6.8, 10% glycerol, 0.02% Bromphenolblau). Cell lysates were boiled at 100°C for 8 min and subjected to 13% SDS-PAGE with the antibody against Caspase-3 (1:800, CST, 9662S), and 10% SDS-PAGE with the antibody against GSDME (1:1000, Abcam, ab215191). Actin (1:1000, HuaBio, ET1701-80) and H3 (1:1000, Proteintech, 17168-1-AP) were used as the internal reference. All the original Western blot data were presented in (Sup. Fig. 3).
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