The cells were lysed and homogenized in RIPA buffer supplemented with protease inhibitors. Protein concentration was determined using a Bicinchoninic Acid Protein Quantification kit (CWBIO, Beijing, China), according to the manufacturer's instructions. Total proteins (50 µg) were loaded on a 12% SDS-PAGE gel, followed by electrophoresis and transfer onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). The sources and dilution ratios of primary antibodies were as follows: GAPDH (1:2,000; cat no. CW0100A; CWBIO), sex determining region Y-box 2 (Sox2; 1:500; EMD Millipore), octamer-binding transcription factor 4 (Oct4; 1:200; cat no. sc-101534), Nanog (1:500; cat no. sc-293121) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and Spalt-like transcription factor 4 (Sall4; 1:500; cat no. ab31968; Abcam, Cambridge, UK). All primary antibodies were incubated overnight at 4°C. The goat-anti-rabbit IgG (for Sox2) and goat-anti-mouse IgG (for GAPDH, Oct4, Nanog and Sall4) horseradish peroxidase-conjugated secondary antibodies were purchased from CWBIO (dilution 1:2,000; cat nos. CW0103 and CW0102, respectively). All secondary antibodies were incubated for 1 h at 37°C. The antigen-antibody complex was visualized using Immobilon Western Chemiluminescent HRP substrate (EMD Millipore).
Gapdh
Manufactured by CWBIO
Sourced in China, United States
GAPDH is a critical enzyme involved in the glycolytic pathway, responsible for the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. It plays a fundamental role in cellular energy production and is commonly used as a reference gene or loading control in various molecular biology and biochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Bca protein assay kit, by CWBIO (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
E cadherin, by Cell Signaling Technology (4 mentions)
Ripa lysis buffer, by CWBIO (3 mentions)
Goat anti mouse igg hrp, by CWBIO (3 mentions)
Goat anti rabbit igg hrp, by CWBIO (3 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Proliferating cell nuclear antigen (pcna), by Bioworld Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
C digit blot scanner, by LI COR (2 mentions)
Runx2, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bca assay, by Beyotime (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Bcl 2, by Bioworld Technology (2 mentions)
Bcl2 associated x (bax), by Bioworld Technology (2 mentions)
44 protocols using gapdh
1
Pluripotency Marker Analysis in Cells
2
mRNA Expression Evaluation via Real-Time PCR
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Evaluation of mRNA expression was performed by using real-time polymerase chain reaction as previously described [28 (link)]. Total RNA was extracted from the colon tissue samples by using extraction kits (CWbio, Beijing, China). After reverse transcription, PCR amplification was performed using the TRIzol method (TRIzol reagent; Invitrogen Life Technologies, Carlsbad, CA, USA). The real-time PCR primer sequences for target genes were as follows: 5′-GCGGCTAGTCCTAACTGTCC-3′/5′-GAATTGGGAAGCCTTGCTGC-3′ for IP10, 5′-TCACTTCCTCTGTTCACGGC-3′/5′-AGGAGGCTGTAGAGGACTGG-3′ for CXCR3, 5′-CAGACACCTTTGCACTTGGC-3′/5′-CTTGAGTAGGACCCCGAGGA-3′ for NF-κB p65, and 5′-CCCATCTATGAGGGTTACGC-3′/5′-TTTAATGTCACGCACGATTTC-3′ for GAPDH (CWbio, Beijing, China). For real-time PCR, the cycling conditions were 95°C for 10 min and 45 × (95°C for 10 s, 59°C for 60 s), followed by a melting curve analysis-based assay with conditions of 95°C for 15 s and 72°C for 15 s and increase in temperature to 95°C for 15 s. Relative expression was assessed by calculating the expression relative to that of GAPDH by using the 2−ΔΔCt method.
3
Western Blot Analysis of Exosomal Markers and Apoptotic Proteins
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hucMSC-exosomes or H9C2(2-1) cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and phenylmethanesulfonyl fluoride (PMSF). The protein concentration was determined using the BCA protein assay kit. Equal quantities of protein were loaded and run on 12% SDS gels and then transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk for 1 h and incubated with diluted primary antibodies [CD9 (1 : 500; Bioworld), CD63 (1 : 300; SAB), Bcl-2 (1 : 500; Bioworld), Bax (1 : 500; Bioworld), and GAPDH (1 : 2,000; CWBIO)] at 4°C overnight. The membranes were incubated in goat anti-rabbit or anti-mouse antibodies (1 : 2,000; Bioworld) at 37°C for 1 h. The target proteins were detected using the Luminata Crescendo Western HRP substrate (Millipore, USA) and the results were analyzed by AlphaView SA.
4
Western Blot Analysis of Myogenic Regulators
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Tissue proteins were lysed in RIPA lysis buffer (Cwbio Inc., Beijing, China) and a protease inhibitor cocktail (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein extracts were electrophoresed on 15% SDS-PAGE gels and transferred onto PVDF membranes (GE Healthcare Inc., Menlo Park, California, USA). Membranes were blocked with 5% skimmed milk for 2 hr at room temperature and incubated overnight at 4°C with primary antibodies against FBXO32 (RRID: AB_2246982 , Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000 dilution), Myod (RRID: AB_631992 , Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:500 dilution), MyoG (RRID: AB_784707 , Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000 dilution), MYF5 (RRID: AB_10975611 , Abcam Inc., Cambridge, MA, USA, 1:1000 dilution) and GAPDH (RRID: AB_2651183 , CwBio Inc., Beijing, China, 1:1000 dilution). The detection was carried out with horseradish peroxidase (HRP)–conjugated goat anti-rabbit or anti-mouse secondary antibody (ZSGB-Bio Inc., Beijing, China, 1:5000 dilution) and visualized with an enhanced chemoluminescence (ECL) kit (Thermo Fisher Scientific Inc., Rockford, IL, USA), according to the manufacturer's protocol. Each experiment was performed in triplicate.
5
Antibody-based Protein Detection and Inhibitor Assessment
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The antibodies used targeted the following proteins (dilutions used are included):
GAPDH (CW0101: immunoblotting, 1:1000) from CWBIOTECH; F-actin (40734ES75: immunofluorescence, 1:100) from YEASEN; U1A (10212: immunoblotting, 1:500) from Proteintech; CD63 (67605-1-Ig: immunoblotting, 1:5000) from Proteintech; CD9 (60232-1-Ig: immunoblotting, 1:5000) from Proteintech; TSG101 (14497-1-AP: immunoblotting, 1:1000) from Proteintech; GPX4 (67763-1-Ig: immunoblotting, 1:5000; IHC, 1:1000) from Proteintech; and DHODH (14877-1-AP: immunoblotting, 1:2000; IHC, 1:100) from Proteintech.
The inhibitors used are as follows:
Sorafenib (HY-10201: 10 μM for the in vitro assay and 30 mg/kg for the in vivo assay) and Ferrostatin-1 (HY-100579: 60 nM for the in vitro assay), and Ferrostatin-1 (HY-100579: 5 mg/kg, intraperitoneal injection for the in vivo assay) were obtained from MedChem Express.
GAPDH (CW0101: immunoblotting, 1:1000) from CWBIOTECH; F-actin (40734ES75: immunofluorescence, 1:100) from YEASEN; U1A (10212: immunoblotting, 1:500) from Proteintech; CD63 (67605-1-Ig: immunoblotting, 1:5000) from Proteintech; CD9 (60232-1-Ig: immunoblotting, 1:5000) from Proteintech; TSG101 (14497-1-AP: immunoblotting, 1:1000) from Proteintech; GPX4 (67763-1-Ig: immunoblotting, 1:5000; IHC, 1:1000) from Proteintech; and DHODH (14877-1-AP: immunoblotting, 1:2000; IHC, 1:100) from Proteintech.
The inhibitors used are as follows:
Sorafenib (HY-10201: 10 μM for the in vitro assay and 30 mg/kg for the in vivo assay) and Ferrostatin-1 (HY-100579: 60 nM for the in vitro assay), and Ferrostatin-1 (HY-100579: 5 mg/kg, intraperitoneal injection for the in vivo assay) were obtained from MedChem Express.
6
Liver Tissue Protein Analysis
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The protein concentration was determined in the supernatant of liver tissue. The samples with equal amounts of protein were analyzed for Phospho-Tyr705-STAT3 (Cell signaling Technology, Boston, MA), Phospho-217-C/EBPβ (Santa Cruz Biotechnologies, CA, USA), PGC-1α (Abcam plc, Cambridge, UK), Phospho-Ser32-IқBα (Cell signaling Technology, Boston, MA), PPARα (Santa Cruz Biotechnologies, CA, USA), ABCG5 (Santa Cruz Biotechnologies, CA, USA), ABCG8 (Novus Biological, Littleton, USA), CYP7A1 (Abcam plc, Cambridge, UK), CYP19A1 (Abcam plc, Cambridge, UK), β-actin (Abgent, San Diego, CA, USA) and GAPDH (CWBIO, Beijing, China) by SDS-PAGE and Western blotting.
7
Protein Extraction and Immunoblotting Analysis
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Proteins were extracted from cells on ice with RIPA lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich). The subsequent Western blotting analysis was performed using standard procedure. The primary antibodies used were listed as follows: FAT10 (Merck Millipore, Billerica, MA, USA); SIRT1 (Cell Signaling Technology); α-SMA (Protein Tech, China); fibronectin (ProteinTech Group, Inc.); COL1A1 (Cell Signaling Technology); COL3A1 (Cell Signaling Technology); GAPDH (CWBIO, Beijing, China).
8
Protein Expression Analysis of GC-MSCs
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Pretreated GC-MSCs were homogenized and lysed in RIPA buffer supplemented with proteinase inhibitors. Equal amounts of a total protein were loaded and separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Following electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane, blocked in 5% (w/v) fat-free milk, and incubated with the primary antibodies. The primary antibodies used were: β-TrCP (CST, USA); p-NFκB (CST, USA); t-NFκB (CST, USA); IκBα (CST, USA); GAPDH (CWBIO, China); goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP (CWBIO, China).
9
Quantification of Angiotensin Receptor Proteins
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Extracts of rat tissue were collected using tissue lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) plus 1 mM phenylmethylsulfonyl fluoride. Total protein was quantified using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with PBS and Tween 20 containing 5% non-fat dry milk to prevent nonspecific antibody binding. Equal protein loading was determined using specific antibodies for the antigens of GAPDH (CWBiotech, Beijing, China), ACE1, AT1R and AT2R (Abcam, Cambridge, MA, USA), washed, and incubated with an appropriate IRDye800-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA). Specific immunofluorescence bands were detected and analyzed using the Odyssey infrared imaging system and software (LI-COR Biosciences, Lincoln, NE, USA).
10
Western Blot Analysis of VSMCs
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VSMCs at the logarithmic stage were lysated in a radioimmune precipitation assay buffer (Solarbio, Beijing, PR China) containing 1% phenylmethanesulfonyl fluoride as proteinase inhibitor. After precipitation in 13,000 g for 10 min, the supernatant containing proteins were collected and followed by denaturing in 100 °C with bromophenol blue. The protein samples were loaded to an 8% SDS-PAGE gel for electrophoresis to separate the proteins, and then transferred to the PVDF membranes. The membranes were blocked in 5% fat free milk to avoid non-specific bindings and incubated in primary antibodies against αSMA (BOSTER, Wuhan, PR China), SM22 (Abcam, Cambridge, UK), calponin (BOSTER, Wuhan, PR China), PCNA (Abcam, Cambridge, UK) and GAPDH (CWBIO, Beijing, PR China), followed by incubating in the corresponding secondary antibodies. The membranes were illuminated and photographed in the ChemiDoc imaging system (Bio-Rad). The bands were quantified by NIH imageJ software and the abundance level of proteins was normalized by GAPDH. The protein of interest/GAPDH ratio was shown as mean ± S.D from three separated experiments and p value < 0.05 was considered significant by student’s t test.
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