Cells were processed for western blotting, as described previously (Brouxhon et al., 2007 (link)). Briefly, samples were electrophoretically separated using SDS‐PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) nonfat dry milk to block nonspecific binding, and then incubated with TGF‐βRI, TGF‐βRII, Sox2 and β‐actin (Santa Cruz Biotechnology), Oct4 (Chemicon), Nestin (Novus Biologicals), Nanog, TGF‐βRIII, phospho‐specific Smad2, Smad3, Smad2, Smad3, and Smad4 (Cell signaling). Proteins were detected using appropriate HRP‐conjugated secondary antibodies (Santa Cruz Biotechnology). Immunoreactive bands were detected using the Clarity MaxTM Western ECL Substrate (Bio‐Rad), and visualized using the ChemiDoc MP Imaging System (Bio‐Rad). Western blot signals were normalized to β‐actin, using NIH Scion Image. Results are presented as fold increase relative to controls in triplicate experiments.
Tgf βriii
Manufactured by Cell Signaling Technology
TGF‐βRIII is a laboratory product offered by Cell Signaling Technology. It is a type III transforming growth factor-beta receptor that functions as a co-receptor for the TGF-beta signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Nanog, by Cell Signaling Technology (1 mentions)
Nestin, by Novus Biologicals (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Smad4, by Cell Signaling Technology (1 mentions)
Clarity maxtm western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Tgf β1, by Cell Signaling Technology (1 mentions)
P smad2 3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
2 protocols using tgf βriii
1
Western Blot Analysis of Stem Cell Markers
2
Protein Extraction and Western Blot Analysis
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Protein samples were extracted from the heart tissue as previously described [23, 24] . After grinding frozen tissue with RIPA lysis buffer (Beyotime, Jiangsu, China), cells were lysed in standard lysis buffer. After boiling the samples for 5 min, the protein samples were fractionated by SDS-PAGE (10% -15% polyacrylamide gels). Primary antibodies were used to detect TGFβRIII (Cell Signaling, Beverly, MA), p-smad2/3 (Cell Signaling, Beverly, MA), and TGF-β1 (Cell Signaling, Beverly, MA). Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area × OD) for each group and normalizing to GAPDH (anti-GAPDH antibody from Kangcheng Inc., Shanghai, China) as an internal control.
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