Protein extraction and Western blot analysis were performed as previously described (35 (link)). The antibodies: PTPN14 (1:1000 dilution), pYAP (s127, 1:1000 dilution), total YAP (1:1000 dilution), c-parp/parp (1:1000) and GAPDH (1:1000 dilution) were purchased from Cell Signaling Technology (Denvers, MA). Polyclonal goat anti-rabbit antibody (Cell Signaling Technology) and Western Blotting Detection System (Millipore) were used for exposure.
Western blotting detection system
Manufactured by Merck Group
The Western Blotting Detection System is a laboratory equipment used for the detection and analysis of proteins in biological samples. It is a versatile tool that employs a combination of electrophoresis, transfer, and immunodetection techniques to identify and quantify specific proteins within a complex mixture.
Automatically generated - may contain errors
Lab products found in correlation
Polyclonal goat anti rabbit antibody, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pyaps127, by Cell Signaling Technology (1 mentions)
Ptpn14, by Cell Signaling Technology (1 mentions)
Total yap, by Cell Signaling Technology (1 mentions)
Gadph antibody, by Abcam (1 mentions)
Anti sirt1 antibody, by Abcam (1 mentions)
Anti il 6 antibody, by Cell Signaling Technology (1 mentions)
Anti tnf α antibody, by Abcam (1 mentions)
Anti p65 antibody, by Abcam (1 mentions)
Inos antibody, by Santa Cruz Biotechnology (1 mentions)
Genegnome hr scanner, by Syngene (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
4 protocols using western blotting detection system
1
Protein Analysis via Western Blot
2
Western Blot Analysis of Inflammatory Markers
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The proteins in cells of each group were extracted and centrifuged at 4 degrees and then added to the protein lysate for cell lysis with RIPA lysis buffer-combined protease inhibitor cocktail at a ratio of 99 : 1. After that, the total protein was quantified with the BCA method.
The proteins were transferred to the PVDF membrane by electrophoresis with 12% SDS polyacrylamide gel at 300mA2h and then sealed at room temperature with 5% skim milk sealant and TBST for 1 h. The primary antibodies were anti-SIRT1 antibody (diluted 1 : 2000, Abcam, catalog no. AB32441), anti-P65 antibody (diluted 1 : 1500, Abcam, catalog no. AB16502), anti-TNF-α antibody (diluted 1 : 500, Abcam, catalog no. AB6671), anti-IL-6 antibody (diluted 1 : 2000, Cell Signaling Technology, catalog no. 5216), iNOS antibody (diluted 1 : 2000, Santa Cruz Biotechnology, catalog no. SC-650), and GADPH antibody (1 : 5000, Abcam, catalog no. ab9485). All these antibodies were diluted with closed solution, incubated at room temperature for 1 h, and washed with PBS buffer 3 times for 5 min. The polyclonal goat anti-rabbit antibody 1 : 5000 (Cell Signaling Technology) diluted in the closed solution was used as the secondary antibody. Then, it was detected with ECL chemiluminescence reagent imaging (Amersham) and western blotting detection system (Millipore).
The proteins were transferred to the PVDF membrane by electrophoresis with 12% SDS polyacrylamide gel at 300mA2h and then sealed at room temperature with 5% skim milk sealant and TBST for 1 h. The primary antibodies were anti-SIRT1 antibody (diluted 1 : 2000, Abcam, catalog no. AB32441), anti-P65 antibody (diluted 1 : 1500, Abcam, catalog no. AB16502), anti-TNF-α antibody (diluted 1 : 500, Abcam, catalog no. AB6671), anti-IL-6 antibody (diluted 1 : 2000, Cell Signaling Technology, catalog no. 5216), iNOS antibody (diluted 1 : 2000, Santa Cruz Biotechnology, catalog no. SC-650), and GADPH antibody (1 : 5000, Abcam, catalog no. ab9485). All these antibodies were diluted with closed solution, incubated at room temperature for 1 h, and washed with PBS buffer 3 times for 5 min. The polyclonal goat anti-rabbit antibody 1 : 5000 (Cell Signaling Technology) diluted in the closed solution was used as the secondary antibody. Then, it was detected with ECL chemiluminescence reagent imaging (Amersham) and western blotting detection system (Millipore).
3
Protein Extraction and Western Blot
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Protein extraction and Western blot analysis were performed as described previously (26) . The antibodies used in this study are listed in Supplementary Table S2. Polyclonal goat anti-rabbit antibody (Cell Signaling Technology) and Western Blotting Detection System (Millipore) were used for exposure.
4
Quantitative Western Blot Analysis of Mouse Renal Proteins
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Mouse renal tissue protein was lysed with chilled RIPA (Beyotime) with 1% Protease Inhibitor Cocktail (Sigma-Aldrich). We used 10% SDS-PAGE to separate protein, which was then transferred to NC membranes. The membrane was blocked with 5% skim milk powder in Tris-buffered saline containing 0.05% Tween 20 (TBST), then incubated with primary antibodies against PGC1α (Abcam), Sirt1 (Santa Cruz), or GAPDH (Santa Cruz), then incubated with secondary antibodies in milk. Chemiluminescence was performed with a Western blotting detection system (Millipore). The chemiluminescent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene).
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