Cell proteins were disintegrated using pre-cooled RIPA lysis buffer (Beyotime, Shanghai, China) and quantified by the BCA method [8 (link),11 (link),13–17 (link)]. Sixty micrograms of protein was subjected to 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore). The membrane was blocked using 5% skim milk powder diluted with TBST at 37°C for 1 h and probed with primary antibodies of ICAM-1 (Santa Cruz), VCAM-I (Santa Cruz), phospho-NF-κB p65/NF-κB p65 (Biosciences), PTEN (Abcam), p-PI3K/PI3K (Abcam), p-AKT/AKT (Abcam) and GAPDH (Beyotime) at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibody (Beyotime) for an additional 1 h at room temperature. The protein expression level was visualized using a chemiluminescence detection reagent (Beyotime) and calculated by Image J software, with GAPDH as the internal reference.
Gapdh
Manufactured by Beyotime
Sourced in China, United States, United Kingdom
GAPDH is a glycolytic enzyme that catalyzes the sixth step of glycolysis, the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. It is a widely used reference gene in molecular biology research.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (41 mentions)
Ripa lysis buffer, by Beyotime (27 mentions)
β actin, by Beyotime (16 mentions)
Ripa buffer, by Beyotime (15 mentions)
Bca protein assay kit, by Beyotime (14 mentions)
Erk1 2, by Cell Signaling Technology (10 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (9 mentions)
E cadherin, by Cell Signaling Technology (7 mentions)
Bcl2 associated x (bax), by Beyotime (7 mentions)
Bcl 2, by Beyotime (7 mentions)
α sma, by Abcam (7 mentions)
Bca kit, by Beyotime (6 mentions)
Vimentin, by Cell Signaling Technology (6 mentions)
Vimentin, by Abcam (6 mentions)
Bcl 2, by Cell Signaling Technology (6 mentions)
P akt, by Cell Signaling Technology (6 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (6 mentions)
N cadherin, by Cell Signaling Technology (5 mentions)
Nitrocellulose membrane, by Merck Group (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
236 protocols using gapdh
1
Protein Expression Analysis in Cells
2
Evaluation of CuSO4-Induced Cytotoxicity
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CuSO4, poly-vinylpyrrolidone, and C6H12O6 were obtained from Shanghai Tian Scientific Co. Ltd. Cell culture medium (Dulbecco's modified eagle’s medium, DMEM) were from Hyclone Laboratories. Fetal bovine serum (FBS) were got from Gibico. Acridine orange, ethidium bromide, FITC-Annexin-V/PI apoptosis assay kit, WST-1 assay kit and Hoechst 33342 were obtained from Beyotime Company of China. Antibody VEGFR2 (Flk-1 [C-1158], sc-504, rabbit polyclonal antibody raised against amino acids 1158–1345 of mouse Flk-1, Santa Cruz Biotechnology, USA). MMP-2 (H-76, sc-10736, rabbit monoclonal antibody against human MMP-2, Santa Cruz Biotechnology, USA). MMP-9 (H-129, sc-10737, rabbit monoclonal antibody against human MMP-9, Santa Cruz Biotechnology, USA). GAPDH (AG019, Primary antibodies used were mouse antibodies specific for the GAPDH, Beyotime Institute of Biotechnology, Jiangsu, China). F-actin (Mouse monoclonal [NH3] to F-actin, Abcam). β-actin (AA128, Primary antibodies used were mouse antibodies specific for the β-actin, Beyotime Institute of Biotechnology, Jiangsu, China). Secondary antibodies goat anti‑mouse (A0216, Beyotime Institute of Biotechnology, Jiangsu, China). Goat anti‑rabbit (A0208, 1:1000, Beyotime Institute of Biotechnology, Jiangsu, China).
3
Profiling Transcription Factors in ILC3s
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The protein level of GAPDH, TCF-1, β-catenin, JunB and NFATc2 in ILC3s was detected by Western blot with the following antibodies: GAPDH (Beyotime, AF2819), TCF-1 (clone C63D9, Cell Signaling Technology, 2203), β-catenin (BD, 610154), JunB (clone C37F9, Thermo Fisher Scientific, PA1-835) and NFATc2 (clone 25A10.D6.D2, Thermo Fisher Scientific, MA1-025). In co-immunoprecipitation (co-IP) experiments, ILC3 pellets were resuspended in cell lysis buffer (Beyotime, P0013) including 1 mM PMSF (Beyotime, ST506). Then protein A + G magnetic beads (Beyotime, P2108), mouse IgG1 isotype control (clone G3A1, Cell Signaling Technology, 5415) and mouse anti-β-catenin (clone D10A8, Cell Signaling Technology, 8480) were used for IP. The IgG1-binding and β-catenin-binding proteins were further identified by Western blot.
4
Western Blot Analysis of Pluripotency Markers
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The cells were lysed with RIPA lysis buffer (Beyotime, Haimen, China) supplemented with protease inhibitors (Roche, Basel, Switzerland), according to the manufacturer’s protocol. Protein concentration was determined using the BCA protein assay kit, following the manufacturer’s instructions. For each well, protein lysate (50 mg) was separated on 6–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime, Haimen, China). The membranes were blocked with 5% dried skimmed milk powder for an hour and incubated in primary antibodies to OCT4 (Boster, Wuhan, China) at 4°C overnight and GAPDH (Beyotime, Haimen, China) at 4°C overnight. Subsequently, the membranes were removed and washed with TBST three times for 5 min, followed by incubation with secondary antibody conjugated to horseradish peroxidase (HRP) (Beyotime, Haimen, China) at 1: 11000 dilution at room temperature for 2 h. GAPDH was used as an internal control. Protein bands were was visualized using an enhanced chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA) with a FluorChem™ FC3 system molecular imager (ProteinSimple, San Jose, California, USA).
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted using Radio-Immunoprecipitation Assay (RIPA) buffer (Beyotime Co. Ltd). A bicinchoninic acid (BCA) kit (Beyotime Co. Ltd) was used to measure protein concentration. Proteins (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene uoride membranes (PVDF; Millipore, Billerica, MA, USA). Antibodies used were RECK, Mortalin, E-cadherin, N-cadherin, p-STAT3 Tyr705 , Ac-STAT3 K685 and vimentin (Cell Signaling Technology, 1: 1000), GAPDH and β-actin (Beyotime Co. Ltd, 1: 500) (Supplementary Table S2).
Densitometric analysis was performed using Image-Pro-Plus 6.0 software, and GAPDH or β-actin served as the internal controls to correct for differences in protein loading.
Densitometric analysis was performed using Image-Pro-Plus 6.0 software, and GAPDH or β-actin served as the internal controls to correct for differences in protein loading.
6
Protein Expression Analysis via Western Blot
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The RIPA buffer (Beyotime) containing protease inhibitor (1%, Sigma) was used to extract total protein from tissue specimens and cells. Western blotting was performed according to a previous study [23 (link)]. The primary antibodies were exhibited as blows: anti-multidrug resistance protein 1 (MDR1) (ab170904, 1:2000), anti-cyclinD1 (ab40754, 1:2000), anti-bcl-2 (ab182858, 1:2000), and anti-GAPDH (ab9484, 1:1000). Goat anti-rabbit IgG (ab97051, 1:1000) was used as a secondary antibody. The immunoblot was visualized via using the enhanced chemiluminescence solution (Beyotime) and GAPDH was used as a loading control. All antibodies were bought from Abcam (Cambridge, MA, USA).
7
Immunoblotting of Apoptosis Regulators
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Immunoblotting was conducted as previously described.22 (link) The primary antibodies used in this study were as follows: anti-poly (ADP-ribose) polymerase 1 (PARP, Abcam, Cambridge, MA, USA), anti-BCL2 apoptosis regulator (Bcl-2, Santa Cruz, Dallas, TX, USA), anti-MCL1 apoptosis regulator (Mcl-1, Cell Signaling Technology, Danvers, MA, USA), anti-BCL2 like 1 (Bcl-xL, Abcam), anti-BCL2 associated X (BAX, Abcam), anti-BCL2 antagonist/killer 1 (BAK, Abcam), anti-phosphorylated AKT serine/threonine kinase 1 (p-AKT, Cell Signaling Technology), anti-phosphorylated mitogen-activated protein kinase 1 (p-ERK, Cell Signaling Technology), anti-phosphorylated mechanistic target of rapamycin kinase (p-mTOR, Cell Signaling Technology), anti-MYC proto-oncogene (c-Myc, Abcam), anti-EIF4E (Abcam) and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Beyotime, Shanghai, China).
8
Western Blot Analysis of Key Osteogenic Regulators
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Whole-cell lysates were prepared on ice using cold RIPA lysis buffer (Beyotime, China) containing protease inhibitor (Thermo Fisher, USA). In brief, equal amounts of proteins (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes. After incubation with the primary antibody at 4°C overnight, they were further immunoblotted with HRP-conjugated antibody at 37°C for 1 h, developed with enhanced chemiluminescence (ECL) substrate (Millipore, USA) and chemiluminescence detection by ChemiDocTM MP Imaging System (Bio-Rad, United Kingdom). Primary antibodies and dilutions used were as follows: p53 (1:1,000; Cell Signaling Technology); p21 (1:2,000; Abcam); Runx2 (1:500; Proteintech); Cbfb (1:200; Santa Cruz Biotechnology); BMP9 (1:200; Santa Cruz Biotechnology); and GAPDH (1:2000; Beyotime Biotechnology).
9
Cardiac Fibrosis Protein Analysis
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Rats were anesthetized with iso urane (3.5%) and sacri ced after receiving HSD for 8 weeks. The hearts were removed, immediately frozen in liquid nitrogen and stored at -70 ℃ until use. Heart tissues or cultured cells were sonicated in RIPA lysis buffer and homogenized. The debris was removed and the supernatant was obtained after centrifugation at 12,000 g for 10 min at 4°C. About 30-50 μg proteins was loaded for electrophoresis, and probed with primary antibodies against collagen I (1:1000; No.14695-1-AP; Proteintech Co., Wuhan, China), α-SMA (1:1000; No.14395-1-AP; Proteintech Co.), TGF-β (1:1000;
No.21898-1-AP; Proteintech Co.). GAPDH (1:1000, AF0006; Beyotime Biotechnology Co., Shanghai, China) was used as internal control. Images were analyzed using the Image-Pro Plus software.
No.21898-1-AP; Proteintech Co.). GAPDH (1:1000, AF0006; Beyotime Biotechnology Co., Shanghai, China) was used as internal control. Images were analyzed using the Image-Pro Plus software.
10
Western Blot Analysis of Cell Signaling Proteins
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TLysis buffer (Cell Signaling Technology, Danfoss, MA, USA) containing phenylmethanesulfonyl fluoride (Beyotime) was used to lyse the stimulated cancer cell lines. Equal amounts of extracts were separated to 10% or 12.5% SDS PAGE gels (Abcam, Cambridge, UK), transferred onto polyvinylidene fluoride membranes (EMD Millipore), followed by incubation with primary antibodies at 4°C overnight. The primary antibodies were anti-CDC6 (ab109315, abcam), Ki-67 (ab245113, abcam), anti-PCNA (ab92552, abcam) and GAPDH (AF0009; Beyotime). HRP-conjugated secondary antibodies were used to incubate the membranes for 1 hour at room temperature, after which they were washed with TBST (PBS with 0.05% Tween20) 6 times. Finally, the visualization of the blot bands was achieved by Find-do ×6 Tanon (Tanon, Shanghai, China).
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