Expression of inflammatory cytokines including interleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) in cardiac tissues was evaluated by western blot. Tissues were extracted from myocardium and then homogenized in 500 µl of lysate. After electrophoresis, trans-membrane and blocking, membrane was incubated at 4°C overnight with rabbit anti-rat IL-6 (Sigma, St. Louis, MO, I5018, SAB4300383, 1 : 1500) or TNF-α (Cell Signaling Technology, Danvers, MA, #3727, 1 : 1500). The membranes were washed 3 times with TBS-T and incubated with goat anti-rabbit IgG HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, #7074, 1 : 4000) at room temperature for 1 h. Protein of interest was detected by chemiluminescence, and optical density (OD) was measured in grey scale images with the Volume Contour method. Glyceraldehyde-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Danvers, MA, #2118, 1 : 8000) was used as a loading control. All the data were presented as relative expression after being normalized to GAPDH.
Glyceraldehyde phosphate dehydrogenase gapdh
Manufactured by Cell Signaling Technology
Sourced in United States
Glyceraldehyde-phosphate dehydrogenase (GAPDH) is an enzyme that catalyzes the sixth step of glycolysis, the metabolic pathway that converts glucose into energy. GAPDH is responsible for the oxidative phosphorylation of glyceraldehyde-3-phosphate, producing 1,3-bisphosphoglycerate and NADH.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 p erk1 2, by Cell Signaling Technology (1 mentions)
Blasticidin s, by Merck Group (1 mentions)
Phospho p65 p p65, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
24 well transwell plate, by Corning (1 mentions)
Dulbecco s modified eagle s medium nutrient mixture f 12 dmem f 12, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Merck Group (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Calcium assay kit, by Nanjing Jiancheng (1 mentions)
Alkaline phosphatase alp activity kit, by Nanjing Jiancheng (1 mentions)
Ecl plus western blotting detection system, by Cytiva (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by Cell Signaling Technology (1 mentions)
Nfatc1, by Abcam (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (1 mentions)
Molecular weight marker, by Bio-Rad (1 mentions)
4 protocols using glyceraldehyde phosphate dehydrogenase gapdh
1
Cardiac Inflammatory Cytokine Expression
2
NF-κB Pathway Regulation Analysis
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Antibodies against p65 (cat.no. 4764), phospho-p65 (p-p65, cat.no. 3033), IκBα (cat.no. 8635), phospho-IκBα (p-IκBα, cat.no. 2859), IκBβ (cat.no. 8635), phospho-IκBβ (p-IκBβ, cat.no. 4921), ERK1/2 (cat.no. 4695), phospho-ERK1/2 (p-ERK1/2, cat.no. 4370), JNK (cat.no. 9252), phospho-JNK (p-JNK1/2, cat.no. 4668) and glyceraldehyde phosphate dehydrogenase (GAPDH) (cat.no. 2118) were purchased from Cell Signaling Technology (Beverly, USA). Antibodies against NIBP (cat.no. 16014-1-AP) were obtained from Proteintech Group (Rosemont, USA). PVDF membrane (cat.no. 162–0181) was obtained from Bio-Rad Laboratories (Hercules, USA). Pierce ECL Western Blotting Substrate (cat.no. 32209) was obtained from Thermo Fisher Scientific (Waltham, USA). Blasticidin S (cat.no. 203351) was purchased from EMD Millipore (Darmstadt, Germany). TNF-α (cat.no. T6674) and Matrigel (cat.no. E1270) were purchased from Sigma-Aldrich Co. (St. Louis, USA). The 24-well transwell plates (cat.no.3422) were obtained from Corning Inc. (Corning, USA).
3
Vascular Smooth Muscle Cell Differentiation
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A7r5 VSMCs were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s Medium, Nutrient Mixture F-12 (DMEM/F12) was obtained from Gibco (Grand Island, USA). CML was acquired from Polypeptide Laboratories (San Diego, USA). Streptozotocin (STZ) was obtained from Sigma–Aldrich Co. LLC (St. Louis, USA). A CML ELISA kit was obtained from Meixuan Biological Science and Technology Co., Ltd (Shanghai, China). A calcium assay kit and alkaline phosphatase (ALP) activity kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A Von Kossa staining kit was purchased from Shunbai Biologicals Inc. (Shanghai, China). An Annexin V-FITC apoptosis detection kit was obtained from Sigma-Aldrich Co. LLC (St. Louis, USA). Glyceraldehyde-phosphate dehydrogenase (GAPDH) was acquired from Cell Signaling Technology, Inc. (Boston, USA). Antibodies against ALP, bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (Runx2) and all secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA).
4
Western Blot Analysis of NFATc1 Expression
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Cells were lysed in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentrations were determined using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was calibrated with molecular weight markers (Bio-Rad, Hercules, CA, USA). NFATc1 (1:500; Abcam, Tokyo, Japan), and glyceraldehyde phosphate dehydrogenase (GAPDH; 1:1 000; Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were each used at a dilution of 1:2 000. Bound antibodies were visualized by chemiluminescence using the ECL Plus Western Blotting Detection System (Amersham, Uppsala, Sweden), and images were analyzed by a Luminescent Image Analyzer (LAS-3000; Fuji Film, Tokyo, Japan).
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