BPC lysed by RIPA Lysis and Extraction Buffer (ThermoFisher Technology, Beijing, China). Protein content was determined by the BCA protein assay reagent (Betotime Biotechnology, China), and 20 μg of each sample was subjected to polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes under 200 mA for 90 min. PVDF membranes were incubated with primary antibody (1:1000 dilution; PAB12542, Abnova Diagnostics, Dongguan, China), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Zymed, San Diego, CA, USA). The membrane was re-probed with a primary antibody against GAPDH (1:3000 dilutions; Santa Cruz Biotech., Santa Cruz, CA, USA) as a control56 (link). The assay was repeated to confirm the result.
Primary antibody against gapdh
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The primary antibody against GAPDH is a laboratory reagent used to detect the presence and quantify the levels of the GAPDH protein in biological samples. GAPDH is a commonly used housekeeping protein that is essential for glycolysis and is often used as a control in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Peroxidase conjugated bovine anti mouse igg secondary antibody, by Jackson ImmunoResearch (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Zymo Research (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Enhanced chemoluminescent autoradiography, by Thermo Fisher Scientific (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Anti myd88, by Santa Cruz Biotechnology (1 mentions)
Anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Anti phospho iκb, by Cell Signaling Technology (1 mentions)
Jc 1 probe, by Beyotime (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
Caspase 3 activity assay kit, by Merck Group (1 mentions)
Anti smad2, by Santa Cruz Biotechnology (1 mentions)
Anti smad3, by Santa Cruz Biotechnology (1 mentions)
Bradford dye binding protein assay, by Bio-Rad (1 mentions)
Primary antibody against trim59, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Calponin 3 h 55 sc 28546, by Santa Cruz Biotechnology (1 mentions)
9 protocols using primary antibody against gapdh
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of p53 Expression
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Western blotting analysis was performed as per the manufacturer’s protocols. Total proteins from human samples were extracted by lysis buffer (Beyotime, Nanjing, China). Protein quality and quantity were determined with the Bradford dye-binding protein kit (Bio-Rad Laboratories, CA, USA). Afterward, a total of 40 μg protein was loaded onto a 10% SDS polyacrylamide gel and transferred to a PVDF membrane. The primary antibodies against P53 were purchased from Cell Signal Technology (Danvers, MA, USA). Primary antibody against GAPDH and secondary antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Immunoreactivity of proteins of each sample was determined by enhanced chemoluminescent autoradiography (Thermo Scientific) using a FluorChem Q system (Proteinsimple, Santa Clara, CA, USA). Blots were quantified using Quantity One (Bio-Rad Laboratories, CA, USA).
3
Western Blot and Immunofluorescence Assays
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Western blot and immunofluorescence assays were conducted as previously described.12 The primary antibody against TRPC3 was purchased from Alomone (Alomone Labs, Jerusalem, Israel), and the primary antibody against GAPDH was purchased from Santa Cruz Biotechnology (Dallas, TX). The primary antibody against phosphorylated PDHE1α (p‐PDHE1α) was purchased from Merck‐Millipore (Darmstadt, Germany). Primary antibodies against PDHE1α and voltage‐dependent anion‐selective channel and fluorescently labeled secondary antibodies were purchased from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)‐labeled secondary antibodies were purchased from Zhongshan Company (Beijing, China).
4
Western Blot Analysis of Inflammatory Signaling
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A Western blot analysis was performed as already described [43 (link)]. Membranes were probed with the following primary antibodies: anti-phospho- IκB (Cell Signaling 2859), anti-MyD88 (Santa Cruz Biotechnologies, sc-74532), and anti-TLR4 (Santa Cruz Biotechnologies, sc-293072) in 1× PBS, 0.1% Tween-20, 5% w/v non-fat dried milk (PMT) at 4 °C overnight [44 (link),45 (link)]. The membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) [46 (link)]. The blots were also incubated with the primary antibody against GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). Signals were detected with an enhanced chemiluminescence detection system reagent according to the manufacturer’s instructions (SuperSignalWest Pico Chemiluminescent Substrate, Pierce, WA, USA) [46 (link)].
5
NBP-Mediated Mitochondrial Regulation and Antioxidant Defense
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NBP was obtained from Shijiazhuang Pharma Group NBP Pharmaceutical with a purity of >99%. The drug was dissolved in DMSO (Sigma-Aldrich, St Louis, MO, USA). The reagents MTT and JC-1 probe were purchased from Beyotime Institute of Biotechnology (Shanghai, People’s Republic of China). Rhodamine-123, 2′,7′-dichlorofluoresceine diacetate (DCF-DA), Mito Tracker Green FM probe and the Caspase-3 Activity Assay kit were obtained from Sigma-Aldrich. ELISA kits to assay superoxide dismutase (SOD), MDA, mitochondrial respiratory chain enzymes activities I–IV and mitochondrial ATPase were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, People’s Republic of China). Primary antibodies against Mfn1, Mfn2, OPA1, Drp1, Fis1, Nrf2, HO-1 and AMPK were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6
Western Blot Analysis of Fibrosis Markers
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Western blot analysis was performed as previously described [46 (link)]. Membranes were probed with one of the following primary antibodies: anti-NOX-4 (PA5-72816), anti-TGFβ1 (Santa Cruz Biotechnology, Heidelberg, Germany, sc-130348), anti-p-Smad3 (cell signaling), anti-p-Smad2 (cell signaling), anti-Smad3 (cell signaling), anti-Smad2 (cell signaling), anti-Col13a1 (Santa Cruz Biotechnology, sc-514601), anti-Col11a1 (Thermo Fisher), in 1 x PBS, 0.1% Tween-20, 5% w/v non-fat dried milk (PMT) at 4 °C overnight [47 (link),48 (link)]. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) [49 (link)]. Blots were also incubated with primary antibody against GAPDH (Santa Cruz Biotechnology). Signals were detected with an enhanced chemiluminescence detection system reagent according to the manufacturer’s instructions (SuperSignalWest Pico Chemiluminescent Substrate, Pierce) [49 (link)].
7
Western Blotting Analysis of TRIM59 Expression
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Western blotting analysis was performed according to the manufacturer’s protocols. Total proteins from both human tissues and cultured cells were extracted with lysis buffer, and the protein quality and quantity were detected by the Bradford dye-binding protein assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 50 μg of protein was loaded onto a 10% SDS polyacrylamide gel, after which PVDF membrane was used to transfer the proteins. The primary antibody against TRIM59 was purchased from Abcam (Cambridge, MA, USA), and primary antibody against GAPDH and secondary antibodies were commercially purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
8
Western Blot Analysis of miR-206 Target
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To validate the target CNN3 of miRNA miR-206, western blotting was used to analyze interaction of miRNA-mRNA at the protein level. PIEC were seeded in 6-well plates, and 48 h after transfection, the cells were harvested for protein extraction. The protein concentration was measured with a BCA protein assay (Pierce Chemical, Rockford, USA) according to the manufacturer’s protocol, and 20 μg of each sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on an SDS-acrylamide gel. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA) and incubated with primary antibody (Calponin 3 (H-55): sc-28546; 1:1000 dilution; Santa Cruz Biotech., Santa Cruz, CA, USA) followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000 dilution; Zymed, San Diego, CA, USA). The membrane was re-probed with a primary antibody against GAPDH (1:3000 dilutions; Santa Cruz Biotech., Santa Cruz, CA, USA) as a control. The assay was repeated to confirm the result.
9
Gastric Cancer Cell Lines Protocol
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The human gastric cancer KATO III, SGC-7901 and AGS cell lines, as well as the 293T cell line as a control, were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The human gastric cancer MKN45 and MKN74 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Primary antibodies against caspase-3 and caspase-9 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The primary antibody against GAPDH and the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
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