Cells and tissues were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% NaDOAc, 0.1% SDS, and 1.0% NP-40) plus Complete Protease Inhibitor Cocktail and phosphatase inhibitors (Roche, South San Francisco, CA, USA). Protein concentrations from cell lysates and tissue lysates were determined by the Bio-Rad method. Proteins were separated by SDS-PAGE (12%) and transferred to PVDF membranes, which were incubated with antibodies against mouse GSDMD (1:1000, ab219800, Abcam), human GSDMD (1:1000, ab210070, Abcam), or β-actin (1:5000, sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by a 1-h incubation with secondary goat anti-rabbit IgG (1:5000, A21109, Thermo Fisher Scientific, Waltham, MA, USA). The signals were developed using the Li-Cor Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
A21109
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A21109 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for use in scientific research and analysis. The core function of this product is to perform a specific task or measurement within a laboratory setting. No further details about the intended use or capabilities of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (5 mentions)
Sc 47778, by Santa Cruz Biotechnology (3 mentions)
A21057, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor and phosphatase inhibitor cocktail, by Roche (2 mentions)
Ab209845, by Abcam (2 mentions)
A21058, by Thermo Fisher Scientific (2 mentions)
G8795, by Merck Group (2 mentions)
Image studio software, by LI COR (2 mentions)
Ab210070, by Abcam (1 mentions)
Ab219800, by Abcam (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Halt phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ab11316, by Abcam (1 mentions)
Ab113692, by Abcam (1 mentions)
Be2011, by EASYBIO (1 mentions)
Ac008, by ABclonal (1 mentions)
11 protocols using a21109
1
Western Blot Analysis of GSDMD
2
Western Blot Analysis of Glutaminase
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Following 4 h treatment, all cells were scraped, washed thrice with ice-cold 1 × PBS with centrifugation at 1,500 rpm at 4 °C, lysed using the RIPA buffer and sonication, as previously69 . Lysates were cleared of debris with centrifugation at 14000 rpm. The Pierce® BCA Assay (Thermo Fisher Scientific, USA) was used to estimate the protein concentration. The 5 × sample-buffer was added into protein (20 μg) from sample and heated to denature the protein. SDS-polyacrylamide gel electrophoresis was performed to separate the denatured proteins and were transferred onto a polyvinyl difluoride (PVDF) membrane for antibody staining and protein detection. The PVDF membrane was incubated overnight with 5% (w/v) nonfat dry milk at 4 °C for blocking. The blocked membrane was probed for proteins using primary antibodies for GLS (rabbit, PA5-35365; Thermo Fisher Scientific) and β-tubulin (mouse, E7; DSHB, University of Iowa). The proteins were detected using the appropriate Alexa Fluor® secondary antibody (anti-rabbit, A-21109 or anti-mouse, A11375; Thermo Fisher Scientific). The blots were imaged at 680 nm for GLS and 790 nm for β-tubulin using the Li-Cor Odyssey infrared imaging System and were quantified using ImageJ.
3
GSDMD Expression in LPS/Nigericin-Treated BMMs
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BMMs were treated with 100 ng/mL LPS for 3 hours, then with 15 μM nigericin for 30 minutes. Extracts from BMMs or bone marrow cells were prepared by lysing cells or cell pellets, respectively, with RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% NaDOAc, 0.1% SDS, and 1.0% NP-40) plus phosphatase inhibitors and Complete Protease Inhibitor Cocktail (Roche, Brighton, MA). Protein concentrations were determined by the Bio-Rad method, and equal amounts of proteins were subjected to SDS-PAGE gels (12%). Proteins were transferred onto nitrocellulose membranes and incubated with GSDMD antibody (1:1,000, ab209845, Abcam, Cambridge, MA) or β-actin (1:5,000, sc-47778, Santa Cruz Biotechnology, Dallas, Texas) overnight at 4°C, followed by a 1-hour incubation with secondary goat anti-mouse IgG (1:5,000, A21058, Thermo Fisher Scientific, Grand Island, NY) or goat anti-rabbit IgG (1:5,000, A21109, Thermo Fisher Scientific), respectively. The results were visualized using Li-Cor Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebraska).
4
GSDMD Protein Detection in Macrophages
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BMDMs and OCs were primed with 100 ng/mL LPS for 3 hours, then treated with 15 μM nigericin for 30 minutes. To collect proteins, cells or tissues were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% NaDOAc, 0.1% SDS, and 1.0% NP-40) plus Complete Protease Inhibitor Cocktail and phosphatase inhibitors (Roche, South San Francisco, CA, USA). Protein concentrations from cell lysates and tissue lysates were determined by Bio-Rad method. Proteins were separated by SDS-PAGE (12%) and transferred to PVDF membrane. Proteins were stained with antibodies against GSDMD (1:1,000, ab209845, Abcam), or β-actin (1:5,000, sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C, followed by a 1-hour incubation with secondary goat anti-rabbit IgG (1:5,000, A21109, Thermo Fisher Scientific, Waltham, MA, USA) or goat anti-mouse IgG (1:5,000, A21058, Thermo Fisher Scientific, Waltham, MA, USA), respectively. The signals were developed using Li-Cor Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
5
Western Blot Analysis of LDHA and AKT
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Frozen tissues were homogenized in RIPA buffer with Halt Protease Inhibitor Cocktail (Thermo Fisher 78430) and Halt Phosphatase Inhibitor (Thermo Fisher 78420). Concentration was measured using DC Protein Assay (Biorad 5000116). Samples were boiled with SDS sample buffer for loading. Protein homogenate (30 µg) in SDS sample buffer was subjected to SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% milk in TBS-T (TBS, 0.1% TWEEN 20). Membranes were incubated overnight at 4 °C with primary antibodies (1:1,000) from Cell Signaling phospho-LDHA (#8176), LDHA (#2012), AKT (#9272), phospho-AKT S473 (#4058). Membranes were washed in TBS-T and incubated for 1 h at room temperature with the secondary antibody (Invitrogen A21109). Fluorescent secondary antibody was detected on Licor Odyssey and analysis of band intensity was performed in ImageJ.
6
Antibody Staining Protocol for Cell Imaging
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Mouse antibody to α‐Tubulin‐FITC (1:400, F2168) was purchased from Sigma (USA). Mouse antibodies to γ‐Tubulin (1:200, ab11316) and TPM3 (1:100, ab113692) were purchased from Abcam (USA). Mouse antibody to FLAG (1:2000, M20008L) was purchased from Abmart (Shanghai, China). Rabbit antibody to MYC (1:1000, BE2011) was purchased from EASYBIO (Beijing, China). Rabbit antibodies to β‐Actin (1:2000, AC026) and β‐Tubulin (1:1000, AC008) were purchased from ABclonal (Wuhan, China). Rabbit antibodies to RNF20 (1: 1000, 21625‐1‐AP) and TPM3 (1:1000, 10737‐1‐AP) were purchased from Proteintech Group (USA). Rabbit antibodies to H2Bub (1: 1000, 5546s), H2B (1: 1000, 12364S) and H3 (1: 1000, 4499S) were purchased from Cell Signaling Technology (USA). Mouse antibody to HEC1 (1: 100, sc‐515510) was purchased from Santa Cruz Biotechnology (USA). Rabbit antibodies to RBBP7 (1:100, bs8620) and UBASH3B (1:100, bs8741) were purchased from Bioworld (Beijing, China). Human antibody to ACA (1: 200, 15–234) was purchased from Antibodiesinc (USA). Goat anti‐rabbit FITC (1:200, ZF‐0311) and goat anti‐mouse TRITC (1:200, ZB‐2305) were purchased from ZSGB‐BIO (Beijing, China). Alexa Fluor 680‐conjugated goat anti‐mouse (1:10000, A21057) and Alexa Fluor 800‐conjugated goat anti‐rabbit (1:10000, A21109) were purchased from Invitrogen (USA).
7
Western Blot and Immunofluorescence Assays
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Antibodies were used against GR (24050-1-AP, Proteintech), phosphorylated GR (serine 211) (4161, Cell Signaling), Rac1 (610651, BD Biosciences), RhoA (ARH03, Cytoskeleton), GAPDH (G8795, Sigma-Aldrich). Secondary antibodies conjugated to Alexa Fluor 680 (A21109, Invitrogen) and Alexa Fluor 800 (A11001, Invitrogen) were used for western blotting. A secondary antibody conjugated to Cy2 (711225152, Jackson immunoresearch laboratories) was used for immunofluorescence.
8
Western Blot Profiling of Histone Modifications
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Cells were lysed in 2% SDS, 100 mM tris-HCl (pH 7.5) and equal amounts of protein (≈30 g) were resolved by SDS-polyacrylamide gel electrophoresis (4-12% Bis-Tris) and blotted onto the PVDF transfer membrane (Merck Millipore). Membranes were stained with Ponceau and washed in PBS-Tween 0.01%. Blots were blocked in 5% non-fat milk in PBS-Tween at RT for 30 min, followed by incubation o/n at 4 °C with the primary antibody (SDMA, Cell Signaling Technology, 13222, 1:1000 dilution; SmD3, Sigma, HPA001170, 1:1000 dilution; α-Tubulin, Cell Signaling Technology, 2125, 1:1000 dilution; Prmt5, Santa Cruz Biotechnology, sc-376937, 1:1000 dilution; Tri-Methyl-Histone H3 K27, Cell Signaling Technology, 9733, 1:1000 dilution, Histone H3, Cell Signaling Technology, 4499, 1:2000 dilution; Ezh2, Cell Signaling Technology, 5246, 1:1000 dilution; Tri-Methyl-Histone H3 K4, Cell Signaling Technology, 9725, 1:1000 dilution; all prepared in blocking buffer) and the with a secondary antibody at 4 °C for 5 h (goat α-rabbit IgG-680; A-21109, Invitrogen, 1:5000 dilution; goat α-mouse IgG-680; A21057, Invitrogen, 1:5000 dilution). Blots were imaged with the Odyssey Infrared Imaging System (LI-COR, NE, USA).
9
Western Blot Protein Extraction and Analysis
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Cells were harvested by trypsinization (attached cell lines) or centrifugation (L1236 cells) and washed once with cold PBS. The cell pellets were resuspended in cold RIPA buffer (20 mM Tris-Cl pH 8.0,1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) supplemented with cOmplete protease inhibitor (Roche 11836145001) and PhosStop (Roche 04906837001), for 30 min on ice with occasional mixing. The samples were sonicated for 5 seconds at 40% amplitude using a Vibra-Cell 75186 machine. The tubes were centrifuged at 14000g, 4°C for 10 minutes and the supernatant was used for protein quantification using the BCA method (Pierce 23225). Samples were mixed with 2X Laemmli buffer and incubated for 10 min at 95°C. Thirty µg of protein was loaded per lane on either 15%, 10% or 4-20% polyacrylamide gels. The nitrocellulose membranes were blocked for 30 min at RT with 5% bovine serum albumin (BSA) dissolved in TBST solution. Antibodies were diluted (1:2000) in TBST with 5% BSA, and incubated overnight at 4°C with gentle shaking. Membranes were washed 3 times with TBST and incubated with fluorochrome conjugated secondary antibodies (Invitrogen, A21109, A21057, or A32735), for one hour at room temperature. Membranes were then washed 3 times with TBST and scanned using a Lycor Odyssey system.
10
Quantifying Iron Homeostasis Protein Levels
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