Total protein of EC and non-tumoral tissues, and TE-1 and KYSE-30 cells was extracted, and the protein concentration was measured by bicinchoninic acid kit (Boster, Hubei, China). After that, the extracted protein was supplemented to the loading buffer, and each well loaded with 30 μg sample. Then, the protein was separated by electrophoresis with 10% polyacrylamide gel (Boster), electroblotted onto the polyvinylidene fluoride membrane, and sealed with 5% BSA. Then primary antibody KLK5 (1:1000, Abcam, Cambridge, UK) and GAPDH (1:2000, Jackson Immuno Research, Grove, Pennsylvania, USA), as well as horseradish peroxidase-labeled secondary antibody (1:500, Jackson Immuno Research) were added for incubation. Images were obtained with Odyssey two-color infrared fluorescence scanning imaging system, and the gray values of target bands were determined by Quantity One image analysis software.
Gapdh
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
GAPDH is an enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate during glycolysis. It is a commonly used internal control and reference gene in molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Polyacrylamide gel, by Boster Bio (4 mentions)
Bicinchoninic acid kit, by Boster Bio (3 mentions)
β actin, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Gapdh, by Abcam (1 mentions)
Ripa lysis buffer, by Bio Basic (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phospho smad2 3, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
15 protocols using gapdh
1
Quantitative Protein Analysis in Tissues
2
Immunoblotting Antibodies for Cell Signaling
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Primary antibodies used in western blots were as follows: rabbit CASPASE-3 (#9662, Cell Signaling), rabbit cleaved CASPASE 3 (#9661, Cell Signaling), rabbit CDC6 (#3387, Cell Signaling), mouse GAPDH (G8140, US Biological, Swampscott, MA), rabbit MCM4 (BD Pharmingen, San Jose, CA), mouse PCNA (sc-56, Santa Cruiz Biotechnology), rabbit and mouse Yap (sc-15407 and sc-101199, Santa Cruz Biotechnology, Santa Cruz; #4912 Cell Signaling, Danvers, MA; #2060, Epitomics, Burlingame, CA). All antibodies were used at 1:1000 except GAPDH which was 1:2000.
Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). HRP-conjugated Donkey anti-rabbit IgG (711–036–152) and HRP-conjugated Donkey anti-mouse IgG (715–035–150) were used at 1:4000 to 1:5000 for western blots.
Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). HRP-conjugated Donkey anti-rabbit IgG (711–036–152) and HRP-conjugated Donkey anti-mouse IgG (715–035–150) were used at 1:4000 to 1:5000 for western blots.
3
Western Blot Analysis of STAT3 and Smad Signaling
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Kidney tissues were homogenized using RIPA lysis buffer (BioBasic) containing protease inhibitor cocktail (ThermoFisher, Waltham, MA, USA) and PhosSTOP™ phosphatase inhibitor (Roche, Basel, Switzerland). Homogenates were centrifuged at 20,000× g for 30 min to collect the supernatant and protein contents were measured by BCA assay (ThermoFisher, Waltham, MA, USA). Tris-glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted to separate the equal amounts of protein lysate. Then, proteins were electroblotted onto polyvinylidene difluoride membranes (Roche), blocked, and incubated with rabbit or mouse monoclonal antibodies against STAT3 (Proteintech, Rosemont, IL, USA), phospho-STAT3, Smad2/3, phospho-Smad2/3 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Abcam, Cambridge, UK) at 4 °C for 12 h, rinsed and incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at 4 °C for 1 h. The Western blot image was visualized by the HRP-substrate and UVP Imaging System, quantified using the VisionWorks software version 8.21, and normalized to GAPDH.
4
Quantifying TLR4 Signaling Pathway
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The protein expression levels of molecules involved in TLR4-related signaling pathways in the study were determined by western blotting analysis, according to the standard manufacturer’s protocol. The antibodies of mouse monoclonal ApoA5, rabbit monoclonal toll-like receptor 4 (TLR4), mouse monoclonal myeloid differentiation factor 88 (MyD88), rabbit monoclonal nuclear factor kappa B (NF-κB) p65 were all purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), and the antibody of mouse polyclonal GAPDH was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The primary antibodies were all diluted at 1:1000, and the secondary antibody was diluted at 1: 3000. Immunodetection was performed with the ECL-Plus kit (Pierce Biotechnology, USA), and immunoblot signals were quantified using Quantity One Software (Bio-Rad).
5
Western Blot Analysis of Protein Markers
6
Quantitative Protein Expression Analysis
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Total protein of tissues and cells was extracted, of which the concentration was measured by bicinchoninic acid kit (Boster, Wuhan, China). Boiled (30 μg/well) with the loading buffer at 95°C, protein samples were isolated by 10% polyacrylamide gel (Boster), electroblotted onto polyvinylidene fluoride membrane, and sealed in 5% bovine serum albumin. Afterwards, the membrane was probed with primary antibodies Bax, Bcl-2, ERRFI1 (1 : 1000, Abcam, Cambridge, UK) and GAPDH (1 : 2000, Jackson Immuno Research, Pennsylvania, USA), and peroxidase-labeled secondary antibody (1 : 500, Jackson Immuno Research). Processed by the Odyssey dual-color infrared fluorescence scanning imaging system, the protein bands were assessed by Quantity One image analysis software to measure the gray value. The ratio of gray value in each target band to that in internal control band was measured.
7
Western Blot Analysis of FCoV Proteins
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The cell lysates were sonicated and centrifuged at 10,000 × g for 10 min at 4 °C to remove cell debris. The supernatants were loaded onto a 10% SDS-PAGE gel (Bio-Rad) with 6X reducing buffer (T-Pro Biotechnology), and the proteins were transferred onto NC membranes (Bio-Rad). The membranes were blocked with 5% skim milk (BD Biosciences). To analyze the FCoV N protein, the mouse anti-FCoV N protein mAb (1:2000, Bio-Rad MCA2194) was used as a primary antibody. For analyzing the FCoV S protein, the cat serum (#F108215) obtained from clinical cases (Ethical permission ID: NTU-109-EL-00088) was used as a primary antibody (1:500 diluted). GAPDH (1:5000, ThermoFisher AM4300) was used as the loading control antibody. Goat anti-mouse IgG HRP conjugate (1:2000, Jackson ImmunoResearch) was used as the secondary antibody for N protein and GAPDH analysis. Goat anti-cat IgG HRP conjugate (1:2000, Bethyl Laboratories A20-120P) was used as the secondary antibody for S protein analysis. The protein signals were developed using an ECL detection kit (Bio-Rad) and visualized using the ChemiDoc XRS + System and Image Lab Software (Bio-Rad).
8
Western Blot Analysis of Synaptic Proteins
9
Protein Extraction and Western Blot Analysis
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The total protein was extracted and the concentration was detected by a bicinchoninic acid (BCA) kit (Wuhan Boster Biological Technology, Hubei, China). The extracted protein was added with loading buffer (30 μg/well) and boiled at 95°C for 10 min, and then conducted with electrophoretic separation by 10% polyacrylamide gel (Wuhan Boster Biological Technology, Hubei, China) and transferred onto polyvinylidene fluoride (PVDF) transmembranes and fixed by 5% bovine serum albumin (BSA) for 1 h. The protein was added with primary antibodies Ki-67 (1:1,000, Abcam, Cambridge, MA, USA), cyclin D1, cleaved caspase-3, Bax, Bcl-2, PTEN (all 1:1,000 and from Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pan-Ago antibody, clone 2A8 (1:1,000, Millipore, MA, USA), and GAPDH (1:2,000, Jackson ImmunoResearch, PA, USA), incubated at 4°C for 24–48 h, and placed in secondary antibody, which was marked by horseradish peroxidase (1:500, Jackson ImmunoResearch, PA, USA) and then incubated for 1 h. The images were obtained by an Odyssey two-color infrared fluorescence scanning imaging system. The gray value was analyzed by Quantity One imaging analysis software.
10
Western Blot Analysis of Cellular Proteins
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Proteins of cells or tissues were extracted by RIPA buffer. All protein samples were subjected to 5 ng/μl and immunoblot assay with the indicated antibodies. Hsp90 (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Jackson, West Grove, PA, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) were used as internal controls. Anti-HCN2 (Alomone, Jerusalem, Israel) and anti-TSHR (Abcam, Cambridge, UK) were used in Western blot experiments. The representative blot bands were repeated at least three times.
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