Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with a phosphatase inhibitor cocktail and protease inhibitor. After measuring the concentration of the extracted proteins with a BCA Protein Assay kit (Beyotime), equal amounts of protein were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% skim milk. After 1 hr of blocking, the membranes were incubated at 4°C overnight with the following primary antibodies: anti-NETO2 (1:200; Abcam), anti-N-cadherin (1:1,000; CST), anti-E-cadherin (1:1,000; CST), anti-vimentin (1:1,000; CST), STAT3 (1:1,000; CST), phospho-STAT3 (p-STAT3) (Tyr705) (1:1,000; CST), Cyclin D1 (1:1,000; CST) and the internal control anti-GAPDH (1:5,000; Abways). After being washed in TBST three times, the membranes were incubated for 1 hr at room temperature with a secondary antibody (goat anti-mouse/anti-rabbit IgG-HRP; 1:2,000, Abcam). The bands were visualized with a chemiluminescent substrate enhanced chemiluminescence kit according to the manufacturer’s instruction.
Anti gapdh
Manufactured by Abways
Sourced in China
Anti-GAPDH is a laboratory reagent used for the detection and quantification of the enzyme GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) in biological samples. GAPDH is a widely used internal control and reference protein in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti cyclin a1, by Santa Cruz Biotechnology (1 mentions)
Ecl plus kit, by ZSGB-BIO (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Ab0037, by Abcam (1 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
Ae005, by ABclonal (1 mentions)
Nickel chloride hexahydrate, by Maclin Biochemical Technology (1 mentions)
Cobalt chloride hexahydrate, by Maclin Biochemical Technology (1 mentions)
Manganese chloride tetrahydrate, by Maclin Biochemical Technology (1 mentions)
Iron 2 chloride tetrahydrate, by Maclin Biochemical Technology (1 mentions)
Sodium tungstate dihydrate, by Maclin Biochemical Technology (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Maclin Biochemical Technology (1 mentions)
Ros detection kit s0033s, by Beyotime (1 mentions)
Ecl substrate kit, by Beyotime (1 mentions)
12 protocols using anti gapdh
1
Western Blot Analysis of EMT Markers
2
Western Blotting for Protein Expression
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Tissue and cell samples were lysed in lysis buffer (PMSF: RIPA, 1:100; Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were quantified using the bicinchoninic acid (BCA) protein assay method, and 20 µg protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking in 5% skimmed milk in Tris-buffered saline Tween-20 (TBST) for 2 h, the membranes were incubated with the primary antibodies overnight at 4°C [anti-FoxM1 (1:1,000; Bioworld Technology), anti-GAPDH (1:8,000; Abways Technology, Wynne, AR, USA), anti-proliferating cell nuclear antigen (PCNA) (#ab2426, 1:2,000; Abcam), anti-cyclin A1 (#sc-751; 1:300; Santa Cruz Biotechnology), anti-E-cadherin (#RLT1453, 1:300; Suzhou Ruiying Biological Technology, Co., Ltd., Jiaozuo, China) and anti-vimentin (#RLT4879, 1:300; Suzhou Ruiying Biological Technology)], followed by incubation for 1 h with the corresponding secondary antibody (1:1,000) at room temperature. An ECL Plus kit (ZSbio, Beijing, China) was used to detect the immunoreactive bands.
3
Immunoblotting for Synaptic Protein Analysis
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Samples were lysed in TEN buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% NP-40, supplemented with protease inhibitors and phosphatase inhibitors. Protein concentration was determined by BCA assay (BCA Protein Assay Kit, Thermo Fisher Scientific). Equal amounts of protein samples were subjected to SDS-polyacrylamide gel electrophoresis and PVDF membrane transfer. Proteins were identified by incubating with indicated primary antibodies and then with appropriate HRP-conjugated secondary antibodies. Protein band intensities were determined using the Image J software [45 (link)]. Antibodies used were: anti-GAPDH (Abways, ab0037), anti-PSD93 (Abcam, ab151721), anti-PSD95 (Cell Signaling Technology, 3450 S), anti-Akt (Cell Signaling Technology, 9272 S), anti-phosphorylated Akt (Cell Signaling Technology, 9271 S), and HRP-conjugated secondary antibodies (Thermo Fisher Scientific, 31430 and 31460).
4
Quantitative Protein Analysis via Western Blot
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Proteins were extracted with RIPA buffer (Beyotime Biotechnology, China)) containing protease and phosphatase inhibitors (Beyotime Biotechnology, China)) and were quantified using Rapid BCA Protein Assay Kit (Thermo Fisher, USA) according to the manufacturer’s instructions. Western blot analysis was performed as previously described19 (link). Briefly, 40 μg lysate was loaded onto SDS-PAGE gels, blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA), and incubated with antibodies. The primary antibodies used in this work including anti-IRF4 (Proteintech, 11247-2-AP, dilution 1:1000), anti-FSTL1 (Abcam, ab71548, dilution 1:1000), anti-β-Tubulin (Abclonal, AC008, dilution 1:5000), anti-GAPDH (Abways, AB0037, dilution 1:5000), anti-flag (Abclonal, AE005, dilution 1:2000).
5
Metal Chloride Catalyzed Oxidation Assay
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Iron(II) chloride tetrahydrate (FeCl2·4H2O), cobalt chloride hexahydrate (CoCl2·6H2O), manganese chloride tetrahydrate (MnCl2·4H2O), nickel chloride hexahydrate (NiCl2·6H2O), Sodium tungstate dihydrate (Na2WO4·2H2O), hexadecyl trimethyl ammonium Bromide (CTAB), 1,3-diphenylisobenzofuran (DPBF) and 3,3,5,5-Tetramethylbenzidine (TMB) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). All chemicals were of analytical grade and used without further purification.
The ROS detection kit (S0033S) and propidium iodide (PI) (ST511) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The primary antibodies, Anti-GAPDH and anti-AID, were purchased from Abways Technology Co., Ltd. (Shanghai, China). and North American ImmunoWay Biotechnology Company (TX, USA). The ECL Substrate Kit (P0018FS) was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China).
Human DLBCL SU-DHL-4 and OCI-LY19 cell lines were purchased from BeNa Culture Collection (BeNa, #BNCC340176, #BNCC338225) (Suzhou, China).
The ROS detection kit (S0033S) and propidium iodide (PI) (ST511) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The primary antibodies, Anti-GAPDH and anti-AID, were purchased from Abways Technology Co., Ltd. (Shanghai, China). and North American ImmunoWay Biotechnology Company (TX, USA). The ECL Substrate Kit (P0018FS) was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China).
Human DLBCL SU-DHL-4 and OCI-LY19 cell lines were purchased from BeNa Culture Collection (BeNa, #BNCC340176, #BNCC338225) (Suzhou, China).
6
Western Blot Profiling of Cellular Signaling
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Western blotting was carried out using an SDS-PAGE Electrophoresis System. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The primary antibodies for this experiment were as follows: anti-GAPDH (AB2000, Abways, Shanghai, China), anti-Col XV (ab202554, Abcam, Shanghai, China), anti-CHOP (YM3668, Immuno Way, Suzhou, China), anti-GRP78 (ab21685, Abcam, Shanghai, China), anti-IRE1α (ab124945, Abcam, Shanghai, China), anti-IL-6 (ab100712, Abcam, Shanghai, China), anti-MCP1 (ab100712, Abcam, Shanghai, China), anti-IL-1β (ab100712, Abcam, Shanghai, China), anti-CD206 (ab125028, Abcam, Shanghai, China), anti-CD163 (YM6146, Immuno Way, Suzhou, China), anti-TNFα (11948P, Cell signaling, Massachusetts, USA), anti-pFAK (ab81298, Abcam, Shanghai, China), anti-FAK (ab40794, Abcam, Shanghai, China), anti-Integrin β1 (ab24693, Abcam, Shanghai, China), Horseradish peroxidase anti-rabbit (Sigma-Aldrich, Shanghai, China) or anti-goat (Sigma-Aldrich, Shanghai, China) was used as secondary antibody. See Antibody Information Sheet (Appendix A ) for antibody details.
7
Protein Extraction and Western Blot Analysis
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Total protein was isolated from the cultured cell lines using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration in the cell extracts was measured using a bicinchoninic acid protein assay kit (Beyotime Biotechnology) with bovine serum albumin as a standard. Equal amounts of protein (40 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% fat-free milk and then incubated with the following primary antibodies at 4°C overnight: anti-KIF26B (Abcam, Cambridge, UK), anti-GAPDH (Abways, Shanghai, China), anti-Bcl-2 (Cell Signaling Technology, Danvers, MA, USA), anti-Bax (Cell Signaling Technology), anti-cyclin D1 (Cell Signaling Technology), anti-p-Rb (ser780) (Cell Signaling Technology), anti-cleaved caspase-3 (Cell Signaling Technology), anti-E-cadherin (Cell Signaling Technology), anti-N-cadherin (Cell Signaling Technology), anti-Vimentin (Cell Signaling Technology), and anti-Snail (Cell Signaling Technology); then, the membranes were incubated with the secondary detection antibody (Cell Signaling Technology) for 1 h at room temperature. Protein bands were visualized using an enhanced chemilumescent (ECL) reagent (Pierce Biotechnology, Waltham, MA, USA).
8
Cell Lysis and Western Blot Analysis
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Cell were lysed with buffer containing 100 mM Tris-HCl (pH 7.5),150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100 with protease inhibitors (Roche, San Francisco, CA) and phosphatase inhibitors (Santa Cruz, Dallas, TX). Protein concentration was determined using the bicinchoninic acid assay (BCA) (Thermo Fisher, Waltham, MA). Equal amounts of proteins samples (approximately 50 μg) were run on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Burlington, MA). The membranes were blocked in TBST (10 mM Tris-HCl pH = 8, 150 mM NaCl, 0.1% Tween) with 5% BSA and probed with primary antibodies and corresponding fluorescence-conjugated secondary antibodies. Antibodies (Supplementary Table 2 ) used included: anti-GAPDH (Catalog No.: AB0037, 1:10,000 dilution) and anti-HIF1α (Catalog No.: CY5197, 1:1000 dilution), purchased from Abways Technology (Shanghai, China); and anti-LEO1 (Catalog No.: ab75721, 1:100 dilution), purchased from ABCAm (Cambridge, MA). Uncropped images of Figs. 6c , 7d are included as Supplementary Figs. 22 and 23 .
9
Liver Protein Extraction and Western Blot
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RIPA (Beyotime Biotechnology, China) containing 1 mM PMSF (Beyotime Biotechnology, China) was used to homogenize liver tissue and then the mixture was incubated on ice for 30 min. Lysates were then centrifuged for 10 min at 14,000 × g at 4 °C. The concentration of protein in the supernatant was measured by bicinchoninic acid assay (BCA) (Beyotime Biotechnology, China). Equal amounts of protein were subjected to 10% ExpressCast PAGE Gels (Cat No. P2012) (New Cell & Molecular Biotech Co., Ltd, China) and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% BSA at 25 °C for 30 min and incubated with the appropriate primary antibody at 4 °C for 12 h. After washing with TBST, the membrane was incubated with the anti-Rabbit IgG. The anti-p-AMPK (Thr172, 1:800, Cat#2535, Cell Signaling Technology), anti-GAPDH (1:1,000, AB0036, Abways) were used to determine the expression of the corresponding proteins. GAPDH was used as an internal control to ensure equal protein loading. The protein bands were monitored using the Odyssey CLx Imager (Licor, USA). The target proteins were quantified using ImageJ software.
10
Quantitative Analysis of Transcriptional and Protein Regulation
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Total RNA isolated using Trizol reagent (Ambion) was subjected to reverse transcription with Superscript Reverse Transcriptase (Invitrogen). RT-qPCR reactions were performed using SYBR Green Master Mix (Thermo) on 7500 Fast real-time PCR system (Applied Biosystems). rPO was used as a reference gene for all qRT-PCR experiments and analyses. The ΔΔCt method was used for quantification analysis. To obtain whole-cell protein extracts or histones, cells were lysed or extracted as previously described46 (link). Membranes were blotted with the corresponding primary antibodies: anti-PRMT2 (1:1000, LifeSpan BioSciences), anti-STAT3 (1:1000, Abways), anti-p-STAT3 (1:1000, Abways), anti-α-Flag (1:2000, Sigma-Aldrich), anti-PTEN (1:1000, Cell Signaling Technology), anti-AXIN2 (1:500, Absin), anti-Histone H3 (1:4000, Millipore), anti-H3R8me2a (1:1000, Novus Biologicals), anti-H3K4me3 (1:1000, Cell Signaling Technology), anti-H3K27ac (1:1000, Abcam), and anti-GAPDH (1:5000, Abways). After careful washing, bound antibodies were detected with HRP-linked anti-mouse or anti-rabbit IgG (CST), followed by electrochemiluminescence (PerkinElmer) determination. All uncropped WBs can be found in Supplementary Figs. 11 and 12 . Primer sequences and information for antibodies are available in Supplementary Table 2 .
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