MEFs grown on six wells were harvested in lysis buffer containing 25 mM Tris-HCl, pH 7.5, 300 mM NaCl and 1% Triton with protease and phosphatase inhibitors. Tissues were minced by a Dounce homogenizer using 1-2 ml RIPA buffer. A total of 30 µg of protein were separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking of the membranes by 5% dry milk/TTBS, membranes were incubated in following primary antibody solutions: anti-Smurf1 (Novus, 1D7); anti-Smurf2 (Abcam, EP629Y3); anti-Smad1 (Cell Signaling, 9743); anti-Smad2 (Abcam, EP784Y); anti-Smad3 (Abcam, ab28379), anti-Smad5 (Abcam, EP619Y), anti-phospho-Smad1/5 (Cell Signaling, 41D10), anti-phospho-Smad2 (Cell Signaling, 138D4); anti-phospho-Smad3 (Rockland, 600-401-919), GAPDH (Santa Cruz, 0411), HSC70 (Santa Cruz, B-6). Protein detection was carried out using HRP-coupled species-specific secondary antibodies and ECL solution, exposed to Hyperfilm ECL.
Ep784y
Manufactured by Abcam
EP784Y is a monoclonal antibody that recognizes the human estrogen receptor alpha (ER-alpha) protein. It is designed for use in various immunoassay applications, including Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab28379, by Abcam (1 mentions)
Hsc70, by Santa Cruz Biotechnology (1 mentions)
Anti smad1, by Cell Signaling Technology (1 mentions)
Anti smad2, by Abcam (1 mentions)
Anti phospho smad1 5, by Cell Signaling Technology (1 mentions)
Anti smurf1, by Novus Biologicals (1 mentions)
Anti smurf2, by Abcam (1 mentions)
Anti smad5, by Abcam (1 mentions)
Anti phospho smad2, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti smad3, by Abcam (1 mentions)
Ab8978, by Abcam (1 mentions)
Mab374, by Abcam (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
2 protocols using ep784y
1
Quantifying Protein Levels in MEFs
2
Western Blot Analysis Protocol
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Western Blot analysis was performed as described previously (Borden et al., 2019 (link)). Briefly, sample concentrations were determined using the Bicinchoninic assay (BCA) according to manufacturer’s protocol and ran on a Mini-PROTEAN TGX Gels (Bio-rad). Primary antibodies against Acta2 (1:1000, rabbit polyclonal, Abcam, catalog ab5694), GAPDH (1:1000, mouse monoclonal, Millipore Sigma, catalog MAB374), CTGF (1:000, rabbit polyclonal, Abcam, catalog ab6992), P-SMAD2 (1:1000, rabbit polyclonal, Millipore Sigma, catalog ZRB04953), SMAD2 (1:1000, rabbit polyclonal, Abcam, catalog EP784Y), and Vimentin (1:1000, mouse monoclonal, Abcam, catalog ab8978) overnight at 4°C, and incubated with the appropriately conjugated light-sensitive IRDye secondary antibodies (1:5000, LiCOR) for 1 h at room temperature, and visualized.
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