HEK293 cells and mouse fibroblast NIH3T3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human primary lung fibroblast cells (HFL) were established by Dr. Reynold Panettieri’s laboratory from patients with brain-related disease but no history of pulmonary fibrosis40 (link). The cells were frozen at an early passage and cultured for a maximum of fifteen passages. The cells were maintained at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DME/F12, Life Technologies/Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), 100 U/mL penicillin G and 100 μg/mL streptomycin (Life Technologies/Gibco). Smad3 inhibitor SIS3 and p38MAPK inhibitor SB203580 were purchased from (AdooQ BioScience, Irvine, CA, USA). Recombinant human TGF-β1 and the Smad2/3 Antibody Sampler Kit were obtained from Cell Signaling Technology (Danvers, MA, USA). The mouse anti-α-SMA monoclonal antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). The rabbit anti-TGFBR1 antibody was obtained from Merck Millipore (Billerica, MA, USA). The mouse anti-CTGF monoclonal antibody, mouse anti-GAPDH monoclonal antibody were obtained from Proteintech (Chicago, IL, USA). The GAPDH levels served as internal controls.
Anti gapdh mouse monoclonal antibody
Manufactured by Proteintech
Sourced in United States, China
The Anti-GAPDH mouse monoclonal antibody is a laboratory reagent used to detect the presence and quantify the levels of the GAPDH protein in biological samples. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a widely used housekeeping gene and its encoded protein is involved in the glycolytic pathway. This antibody can be utilized in various analytical techniques, such as Western blotting, immunoprecipitation, and immunocytochemistry, to study the expression and localization of GAPDH in cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
Hrp conjugated anti mouse igg, by Proteintech (2 mentions)
Nih3t3 cells, by American Type Culture Collection (1 mentions)
Recombinant human tgf β1, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti α sma antibody, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Adooq (1 mentions)
Smad2 3 antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti met, by Cell Signaling Technology (1 mentions)
Anti limk2, by Abcam (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti lamin b, by Proteintech (1 mentions)
Anti β catenin, by Proteintech (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti c jun, by Cell Signaling Technology (1 mentions)
Zli 9017, by ZSGB-BIO (1 mentions)
25 protocols using anti gapdh mouse monoclonal antibody
1
Investigating Lung Fibroblast Cell Lines
2
Western Blot Analysis of EMT Markers
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WB was performed as previously described34 (link), using monoclonal anti-LIMK2 (1:1000; Abcam, Cambridge, MA, USA) and polyclonal anti-LIMK2 (1:1000; Proteintech, Chicago, IL, USA), anti-E-cadherin, anti-Vimentin, anti-Met, anti-c-myc, anti-c-jun (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-LIMK1, anti-β-catenin, anti-lamin B (1:1000; Proteintech, Chicago, IL, USA). The loading control was a mouse anti-GAPDH monoclonal antibody (1:10000; Proteintech, Chicago, IL, USA).
3
Comprehensive Protein Analysis of Tumor Samples
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WB analysis was performed using anti-METTL14, anti-MFN2, anti-FIS1, anti-DRP1, anti-BAX, anti-BCL2, and anti-Caspase7/cleaved-Caspase7 (26158-1-AP, 12186-1-AP, 10956-1-AP, 12957-1-AP, 50599-2-Ig, 12789-1-AP, 27155-1-AP; 1:1000; Proteintech, USA); anti-LC3A/B and anti-p62 (306019, 380612; 1:1000; ZenBio, China); anti-PARP and anti-cleaved-PARP (9542, 5625; 1:1000; CST, USA); and anti-YTHDC2 (ab220160, 1:1000, Abcam, UK) antibodies. The loading control was a mouse anti-GAPDH monoclonal antibody (60004-1-Ig; 1:10000; Proteintech).
For IHC analysis, consecutive tumor sections with a thickness of 4 μm were prepared. Then, deparaffinization and antigen retrieval were performed following the manufacturer’s instructions. These sections were incubated with anti-Ki-67, anti-MFN2 (27309-1-AP; 12186-1-AP; Proteintech) and anti-Caspase7 (T40049S; Abmart, China) antibodies. After incubated with secondary antibodies (PV-6001, ZSGB-BIO, China), the sections were visualized with a DAB chromogenic agent (ZLI-9017, ZSGB-BIO) and observed under a microscope.
For IHC analysis, consecutive tumor sections with a thickness of 4 μm were prepared. Then, deparaffinization and antigen retrieval were performed following the manufacturer’s instructions. These sections were incubated with anti-Ki-67, anti-MFN2 (27309-1-AP; 12186-1-AP; Proteintech) and anti-Caspase7 (T40049S; Abmart, China) antibodies. After incubated with secondary antibodies (PV-6001, ZSGB-BIO, China), the sections were visualized with a DAB chromogenic agent (ZLI-9017, ZSGB-BIO) and observed under a microscope.
4
Western Blot Analysis of Penis Proteins
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The penis protein levels were analyzed by Western blotting on the Odyssey infrared imaging system (LI-COR, Nebraska, USA). In brief, the protein samples of six rats per group were separated by 6–12% sodium dodecyl sulfate polyacrylamide gel (10–20 μg per lane). Primary antibodies were applied after the proteins were transferred to nitrocellulose membranes; GAPDH was used as the loading control. Signals were obtained in the linear range of detection and quantified with the Odyssey infrared imaging system. The data were analyzed and presented as the relative density of each protein relative to GAPDH. The primary antibodies were rabbit anti-PEDF polyclonal antibody (dilution 1 : 1000, Santa Cruz Biotechnologies, Santa Cruz, CA, USA, sc-25594), rabbit anti-phospho-eNOS polyclonal antibody (dilution 1 : 500, Abcam, Cambridge, UK, ab75639), rabbit anti-phospho-Akt monoclonal antibody (dilution 1 : 1000, cell signaling technology, Danvers, MA, USA, #4060), and mouse anti-GAPDH monoclonal antibody (dilution 1 : 5000, Proteintech, USA, 60004-1-Ig.).
5
Western Blot Analysis of Protein Expression
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Total protein was gathered by RIPA Lysis Buffer. The protein samples were separated by 8% SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with 5% BSA for 2 h, the membranes were incubated overnight with primary antibodies including mouse anti-GAPDH monoclonal antibody (Catalog number: 60,004-1-Ig, proteintech, Wuhan, China) and rabbit anti-COL6A2 monoclonal antibody (Catalog number: ab180855, Abcam, Cambridge, UK) at 4°C. After being washed in TBST for three times, membranes were incubated with HRP-conjugated anti-rabbit (Catalog number: SA00001-2, proteintech, Wuhan, China) or HRP-conjugated anti-mouse (Catalog number: SA00001-1, proteintech, Wuhan, China) secondary antibodies at a 1:5000 dilution at room temperature for 1h. Then, the binding secondary antibody was detected and recorded using the enhanced ECL Western blotting detection system.
6
Protein Extraction and Western Blot
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The treated DEFs were lysed with Radio Immunoprecipitation Assay (RIPA ) lysis buffer (Thermo Fisher, USA) containing 1% phenylmethylsulfonyl fluoride (PMSF , Thermo Fisher, USA) for 30 min on ice. The cell lysates or proteins from the RNA pull-down assay were separated by 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). Five percent skim milk was used to block the membranes for 2 h at room temperature. Mouse anti-flag monoclonal antibody (TransGen Biotech, Beijing, China) at a 1:5,000 dilution, mouse anti-E monoclonal antibody (prepared in our lab) at a 1:2,000 dilution, mouse anti-β-actin monoclonal antibody (Proteintech, Rosemont, IL) at a 1:2,000 dilution or mouse anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL) at a 1:2,000 dilution as the primary antibody was added to the membranes and cultured overnight at 4°C. Horseradish peroxidase (HRP )-conjugated goat anti-mouse IgG (Beyotime, Shanghai, China) at a 1:5,000 dilution was used as the secondary antibody. Finally, the Clarity Western ECL Substrate (Bio-Rad, CA, USA) was used for membrane imaging.
7
Lung Tissue Protein Extraction and Quantification
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The lung tissue homogenate was collected then freeze-thawed repeatedly 3–5 times using liquid nitrogen. After being centrifuged at 5000× g for 7 min, the supernatant was protein extracts. Sample protein concentration was determined with the BCA Protein Assay Kit (Genstar). The supernatants were then divided according to the sample protein concentration. Sample loading buffer was added to the supernatants and boiled for 5 min. Samples were then centrifuged, and the supernatant was removed. Approximately 20 μg of sample supernatant was loaded per lane, separated on a precast polyacrylamide Bis-Tris gel with a 4–12% gradient (Solarbio), and transferred onto a nitrocellulose membrane. The membranes were blocked in 10% milk/TBS-T buffer for 1 h at RT and incubated overnight with the following antibodies: rabbit anti-NLRP6 polyclonal antibody (1:2000, Immunoway), rabbit anti-GSDMD polyclonal antibody (1:5000, Proteintech), and mouse anti-GAPDH monoclonal antibody (1:10,000, Proteintech). Membranes were incubated with fluorescently labeled secondary antibodies (1:10,000, Proteintech) at RT for 1 h. The protein bands were detected with the Odyssey® infrared imaging system (LI-COR Biosciences). The relative expression of each target gene was obtained using the following formula:
relative expression of gene = (density of the gene band)/(density of β-actin band).
relative expression of gene = (density of the gene band)/(density of β-actin band).
8
Antibodies for Protein Detection
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Mouse monoclonal antibodies against Flag-tag and HA-tag, FITC-conjugated goat anti-mouse antibodies were purchased from ABclonal Biotechnology (China). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (H + L) secondary antibodies were purchased from Boster Bioengineering (China). Rabbit anti-DnaJA2 polyclonal antibodies and mouse anti-GAPDH monoclonal antibody were purchased from Proteintech Group (USA). The NS3- and NS5- specific polyclonal antibodies were acquired from GeneTex (USA).
9
Investigating HBx-mediated immune signaling
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The primary antibodies used for this study were as follows: mouse anti-HBx monoclonal antibody (Santa Cruz, USA), rabbit anti-cGAS monoclonal antibody (CST, USA), rabbit anti-STING monoclonal antibody (CST, USA), rabbit anti-IRF3(Phospho-Ser382) monoclonal antibody (CST, USA), mouse anti-IRF3 monoclonal antibody (CST, USA), mouse anti-β-actin monoclonal antibody (Santa Cruz, USA), mouse anti-GAPDH Monoclonal Antibody(Proteintech, USA), rabbit anti-Flag polyclonal antibody (Proteintech, USA), rabbit anti-HA polyclonal antibody (Proteintech, USA), K48-linked ubiquitin antibody(Billerica, MA, USA), DAPI (Santa Cruz, USA) and Lipofectamine 2000 (Invitrogen, USA) were used following the manufacturer's instructions.
10
Western Blot Analysis of Protein Expression
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The remaining rats in each group were perfused with 4% chloral hydrate (1 ml/100 g, P57). Hippocampi were placed in a precooled labeled EP tube and then quickly stored at −80°C. Four rat tissues were taken from each group for protein extraction, and the remaining tissues were frozen for other purposes. Detailed steps of the Western blotting method have been previously described (Ni et al., 2018 (link)).
Briefly, polyvinylidene fluoride membrane blots were incubated with one of the following antibodies: goat anti-ZnT3 (1:1000, Santa Cruz), rabbit anti-GPR39 polyclonal antibody (1:1000, Biorbyt), rat anti- MBP monoclonal antibody (1:2000, Abcam), mouse anti-GAPDH monoclonal antibody (1:5000, Proteintech) or rabbit anti-β-tubulin polyclonal antibody (1:5000, Proteintech) in TBST contain 5% non-fat dry milk overnight at 4°C. Membranes were then incubated with secondary antibodies for approximately 2 h. ECL chemiluminescence kit A and B were mixed in equal volume, and PVDF membranes were immersed in the luminescent liquid for approximately 2 min, and then the film was placed in an automatic developing device for exposure (LAS 4010, GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Grayscale values of each band were analyzed using ImageJ image processing software.
Briefly, polyvinylidene fluoride membrane blots were incubated with one of the following antibodies: goat anti-ZnT3 (1:1000, Santa Cruz), rabbit anti-GPR39 polyclonal antibody (1:1000, Biorbyt), rat anti- MBP monoclonal antibody (1:2000, Abcam), mouse anti-GAPDH monoclonal antibody (1:5000, Proteintech) or rabbit anti-β-tubulin polyclonal antibody (1:5000, Proteintech) in TBST contain 5% non-fat dry milk overnight at 4°C. Membranes were then incubated with secondary antibodies for approximately 2 h. ECL chemiluminescence kit A and B were mixed in equal volume, and PVDF membranes were immersed in the luminescent liquid for approximately 2 min, and then the film was placed in an automatic developing device for exposure (LAS 4010, GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Grayscale values of each band were analyzed using ImageJ image processing software.
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