The antibodies used in this study were as follows: anti-PDK1, anti-phospho-PDK1 (Ser241) (GeneTex); anti-phospho-AKT1/2/3 (Ser 473) and AKT1/2/3, anti-ERK1/2 and anti-phospho-ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology); anti-Caspase 3 (3G2), anti-Caspase 9, anti-PARP, anti-STAT3, anti-phospho-STAT3 (Y705) and (Ser727) (Epitomics); anti-JAK1, anti-phospho-JAK1 (Y1022/1023), anti-Smad2, anti-phospho-Smad2 (Ser467), anti-Smad3, anti-phospho-Smad3 (Ser423/425) (Cell signaling); anti-GAPDH (Sangon Biotech., AB10016); anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Pierce). Detection was performed by using a Chemiluminescent Western detection kit (Cell Signaling).
Anti gapdh
Manufactured by Sangon
Sourced in China, United States
Anti-GAPDH is a primary antibody that specifically recognizes the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein. GAPDH is a widely used housekeeping gene and serves as a loading control in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti p akt, by Abcam (2 mentions)
Anti snail, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Chemiluminescent western detection kit, by Cell Signaling Technology (1 mentions)
Anti interleukin 6, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by R&D Systems (1 mentions)
Western ip cell lysis buffer, by Beyotime (1 mentions)
Ab10016, by Sangon (1 mentions)
Ab108424, by Abcam (1 mentions)
Anti smn, by BD (1 mentions)
Anti cleaved parp, by Abcam (1 mentions)
D110016, by Sangon (1 mentions)
Anti ctgf, by Abcam (1 mentions)
Rna extraction kit, by Sangon (1 mentions)
Anti timp1, by Abcam (1 mentions)
Anti mmp 1, by Thermo Fisher Scientific (1 mentions)
19 protocols using anti gapdh
1
Signaling Pathway Antibody Validation
2
Quantifying Protein Level Changes in Spinal Cord
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To qualify temporal changes in protein levels, Western blotting analysis was used. The spinal cord and DRGs at L4–L6) were quickly removed from deeply anesthetized mice and stored at − 80 °C. Sequential precipitation procedures were used on the tissue samples and were lysed in ice-cold (4 °C) RIPA lysis buffer containing a mixture of protease inhibitor, phosphatase inhibitors, and phenylmethylsulfonyl fluoride (Sigma-Aldrich). The total protein was separated by SDS-PAGE and transferred to PVDF membrane (both from Bio-Rad Laboratories, Hercules, CA, USA). The following primary antibodies were used: anti-Interleukin 6 (1:200; Santa Cruz, Santa Cruz, CA, USA), anti-Tumor necrosis factor alpha (1:200; Santa Cruz), anti-GAPDH (1:1000; Sangon, Shanghai, China). The membranes were then developed by enhanced chemiluminescence reagents with horseradish peroxidase-conjugated secondary antibodies (R&D Systems, Minneapolis, MN, USA). Images were acquired with Tanon chemiluminescence instrument (Shanghai, China). Data were analyzed with the ImageJ. Absolute gray level of each plot is quantified with background subtraction and then normalized with the control plot (GAPDH) for comparison.
3
Western Blot Analysis of AQP4 Protein
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Cells from the various treatment groups were collected and lysed using Western/IP Cell Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China) for 1 h at 0°C. Protein concentration was measured by bicinchoninic acid assay. Equal amounts of extracted protein samples (50 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 2% skimmed milk and TBS-0.1% Tween-20 (TBST). Primary polyclonal antibodies against AQP4 (sc-9888; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted (1:500) in TBST were used. Membranes were incubated with the primary antibodies at 4°C overnight, or with anti-GAPDH [1:1,000; AB10016; Sangon Biotech (Shanghai) Co., Ltd] at 4°C for 2 h. Blots were subsequently incubated with horseradish peroxidase-conjugated rabbit anti-goat IgG secondary antibodies [1:1,000; D111047; Sangon (Shanghai) Biotech Co., Ltd.] for 2 h at 4°C. Protein bands were visualized on X-ray film using enhanced chemiluminescence (luminol chemiluminescence substrate, SF-2; Jiangsu Sunlant Bioengineering Co., Ltd.). Developed films were digitized and semi-quantified by densitometry using a gel imaging system (Smartview-2001 Version 5; Shanghai Furi Science & Technology Co., Ltd., Shanghai, China).
4
Antibody Sources and Cobalt Dichloride
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The primary antibodies used in this study were purchased from the following sources: anti-gemin1 (ab108424, Abcam, United Kingdom), anti-SMN (610646, BD Biosciences, United States), anti-cleaved PARP (ab194217, Abcam, United Kingdom), anti-cleaved caspase-3 (9664, CST, United States), anti-NFκB (8242, CST, United States), anti-phosphor- NFκB (3033, CST, United States), anti-IκBα (4812, CST, United States), anti-phosphor-IκBα (2859, CST, United States), and anti-GAPDH (D110016, Sangon Biotech, China). All secondary antibodies used for immunoblot analysis were from Sangon Biotech. Cobalt dichloride (CoCl2, c8661, United States) was purchased from Sigma-Aldrich.
5
Sprague-Dawley Rat Tissue Analysis
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PrimeScript™ RT Master Mix kit was bought from TaKaRa Co. (Kyoto, Japan). The cell lysis kit, RNA extraction kit, and anti-GAPDH were purchased from Sangon Biotech (China). Anti-TGF-β1, anti-CTGF, and anti-TIMP-1 were obtained from Abcam (United Kingdom). Anti-Smad 2 and anti-Smad 3 were obtained from CST (USA). Anti-MMP-1 was obtained from Thermo Fisher Scientific (USA).
Sprague-Dawley (SD) rats (male, 180–220 g) were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. Experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institute Ethics Committee (TCM-2019-013-E04, Tianjin University of Traditional Chinese Medicine).
Sprague-Dawley (SD) rats (male, 180–220 g) were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. Experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institute Ethics Committee (TCM-2019-013-E04, Tianjin University of Traditional Chinese Medicine).
6
Western Blot Analysis of BV-2 Cell Proteins
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Before harvest, BV-2 cells were washed with cold PBS and then lysed with lysis buffer containing protease inhibitors for 30 min on ice. The samples were centrifuged at 12000 rpm, 4°C for 15 min. Then, the protein concentrations were determined by a BCA protein assay kit (Beyotime Institute of Biotechnology, Haimen, China) as previously described [16 (link)]. Proteins were electrophoresed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad, CA, USA) and transferred electrophoretically to PVDF membranes. Then, the membranes were blocked with 5% skim milk at room temperature (RT) for 1 h and then incubated with primary antibodies overnight at 4°C. Subsequently, membranes were washed and incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. Finally, membranes were washed and detected with enhanced chemiluminescence. Primary antibodies were as follows: anti-GAPDH (1 : 2000; Sangon Biotech), anti-β-actin (1 : 2000; Santa Cruz), anti-REST (1 : 1000; Abcam), anti-MHC II (1 : 1000, Abcam), and anti-Arg1 (1 : 1000; Sigma).
7
Protein Extraction and Western Blot Analysis
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The RIPA lysis buffer (Solarbio, Beijing, China) was used to conduct the purification of the protein of the cells and hUCMSCs-EVs. The concentrations of extracted protein were tested and calculated by a bicinchoninic acid protein quantification kit (Thermo Scientific, 23225). A 40 μg of denatured protein was subjected to 10% SDS polyacrylamide gel, subsequently transferred onto the polyvinylidene fluoride membranes (Millipore Corp, Billerica, United States). And then these polyvinylidene fluoride membranes were incubated with first antibodies, anti-IL-4 (Boster, 10K274), anti-TNF-α (Proteintech, 17590-1-AP), anti-MMP13 (Proteintech, 18165-1-AP), anti-CD63 (Bioss, bs-1523R), anti-TSG101 (Abclonal, A1692), anti-CD81 (Bioss, bs-6934R), anti-CALNXIN (Proteintech, 10427-2-AP), or anti-GAPDH (Sangon Biotech Co., Ltd, China) at 4 °C for 12 h after blocking with 5% milk. The secondary antibody, anti-rabbit IgG (Sangon Biotech Co., Ltd, Shanghai, China), was used at 37 °C for one hour. The expression level of each protein was assessed by the Odyssey CLx imaging systems (Li-COR Biosciences, Lincoln, United States).
8
Western Blotting Analysis of Key Cellular Markers
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Tissues and cells were lysed and analysed via Western blotting as previously described.17 The following primary and secondary antibodies were used: rabbit polyclonal anti‐YAP1 (1:1500, ABclonal), rabbit monoclonal anti‐pYAP1 (1:10000, Abcam), rabbit polyclonal anti‐TAZ (1:1000, LifeSpan Biosciences), mouse monoclonal anti‐GLUT1 (1:500, Santa), mouse monoclonal anti‐E‐cadherin (1:500, Santa), mouse monoclonal anti‐Vimentin (1:1000, Santa), rabbit polyclonal anti‐N‐Cadherin (1:1000, CST), rabbit polyclonal anti‐Fibronectin (1:10000, Abcam) and rabbit polyclonal anti‐GAPDH (1:1000, Sangon Biotech). Horseradish peroxidase–conjugated secondary antibodies were purchased from Cell Signaling Technology (1:2000, CST).
9
Western Blot Analysis of cMYC Protein
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The cells were seeded onto six-well plates at a density of 1 × 106 mL−1 and treated with different concentrations of compounds or DMSO control for 6 h. The cell lysates were prepared using 1 × SDS cell lysis buffer and boiled for 20 min. The total cell lysates were separated by 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The blots were blocked with blocking buffer (5% nonfat milk in TBST) for 30−60 min at room temperature and incubated with anti-cMYC (Cell Signaling Technology, Danvers, MA, USA, Cat#5605S) and anti-GAPDH (Sangon Biotech, Shanghai, China, Cat#D110016) primary antibodies overnight at 4 °C. Then, the blots were probed with HRP-conjugated anti-rabbit (Sangon Biotech, Shanghai, China, Cat#D110058) or anti-mouse (Sangon Biotech, Shanghai, China, Cat#D110087) IgG secondary antibodies for 1 h at room temperature. Following another three washes, the bands were detected with a Amersham Imager 600 imaging system (GE Healthcare, Chicago, IL, USA) using ECL substrate (Share-Bio, Shanghai, China, Cat#sb-wb011).
10
Protein Expression Analysis via Western Blot
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Western blot analysis was performed as previously described (Xu et al., 2013 (link)) with the following primary immunoblotting antibodies: anti-GAPDH (Sango Biotech, China), anti-p-AKT, anti-p-RB, anti-p-FOXO3 (Epitomics, United States), anti-Snail, anti-CCND1 (Cell Signaling Technology, United States), and anti-SP1 (Proteintech Group Inc., United States).
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