The extraction of total protein from cultured cells was performed with RIPA lysis buffer (Solarbio, Beijing, China). The concentration of total protein was measured with the BCA Protein Assay Kit (Beyotime, Shanghai, China). The equivalent amounts of total protein were separated by SDS polyacrylamide gel electrophoresis in a 10% gel and transferred to polyvinylidene difluoride membranes, which were then blocked at room temperature for 2 h in 5% fat-free milk diluted in Tris-buffered saline with 0.1% of Tween 20 (TBST). After overnight incubation at 4°C with primary antibodies in TBST, the membranes were washed with TBST thrice and then incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (sc-516102; 1:5000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 h. The detection of protein signals was carried out by means of an Enhanced Chemiluminescence Detection System (Pierce, Rockford, IL, USA). The primary antibodies used in this study included a mouse anti-human ETS1 monoclonal antibody (sc-55581; 1:1000 dilution; Santa Cruz Biotechnology), and a mouse anti-human GAPDH antibody (sc-47724; 1:1000 dilution; Santa Cruz Biotechnology). GAPDH served as a loading control.
Sc 55581
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-55581 is a laboratory product manufactured by Santa Cruz Biotechnology. It serves as a tool for research purposes, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. More information from the manufacturer would be required to present a concise and accurate description of this product.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg secondary antibody sc 516102, by Santa Cruz Biotechnology (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
Sc 17785, by Santa Cruz Biotechnology (1 mentions)
A31571, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Application suite x software, by Leica (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
Slide 1 0.4 mm ibitreat, by Ibidi (1 mentions)
Sc 20691, by Santa Cruz Biotechnology (1 mentions)
3 protocols using sc 55581
1
Western Blot Analysis of ETS1 Protein
2
Chromatin Immunoprecipitation Protocol
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Chromatin immunoprecipitation was performed using an SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, Danvers, MA, USA). Pre-cleared chromatin was immunoprecipitated with mice monoclonal antibodies against ETS1 (sc-55581, Santa Cruz Biotechnology, Dallas, TX, USA) or MEF2A (sc-17785, Santa Cruz Biotechnology, Dallas, TX, USA), normal rabbit IgG or antibody against acetyl Histone H3 (included in the kit). The total cell chromatin extract was used as input. Protein-DNA crosslinks were precipitated by using ChIP-Grade Protein G Magnetic Beads. And DNA is purified using DNA purification spin columns (included in the kit). Following quantification was undertaken by quantitative PCR analysis with primers listed in Supplementary Table S7 .
3
Quantification of Transcription Factor Expression
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PAEC were cultured on flow slides (µ-Slide I 0.4 mm ibiTreat; Ibidi), washed with PBS, and fixed with ice-cold methanol at −20 °C for 30 min. Methanol was aspirated and the slides were rehydrated with PBS at room temperature for 10 min. After washing with PBS, slides were blocked with 5% normal donkey serum and 2% BSA (Sigma Aldrich) in PBS at room temperature for 1 h. Incubation with primary antibodies targeting KLF4 (1:100, sc20691, Santa Cruz Biotechnology), ATF2 (1:100 sc-242, Santa Cruz Biotechnology) and ETS1 (1:100, sc55581, Santa Cruz Biotechnology) were carried out in the blocking buffer at 4 °C overnight. Secondary antibody incubations were performed using 1:600 dilutions of A-21206 (for KLF4); A-21203 (for ATF2) and A-31571 (for ETS1), all from ThermoFisher Scientific, in the blocking buffer at room temperature for 1 h. Slides were mounted with DAPI Fluoromount-G (DAPI, 4,6-diamidino-2-phenylindole) (SouthernBiotech). Stained slides were imaged using Leica Application Suite X software on a Leica Sp8 (Leica). Quantification of the nuclear fluorescence intensities was performed using ImageJ.
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