Immunoblotting Analysis of Smad and Akt Signaling

Cell lysis, protein extraction, and immunoblotting were performed as described^{83 (link)}. Protein concentrations were quantified using Bradford assays (Bio-Rad) with bovine serum albumin (BSA) as standard, and a SpectraMax M5 microplate reader. Samples were resolved by SDS-PAGE on 4–12% gradient polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and detected by immunoblotting. As primary antibodies, we used rabbit monoclonal antibodies to Smad2, phospho-Smad2 (Ser^465/467), Smad3, Akt, and phospho-Akt (p-Akt^S473) from Cell Signaling. Antibody against phospho-Smad3 (Ser^423/425) was from Abcam, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Sigma. For immunoblotting, immunoreactive bands were visualized using Western Lighting Plus ECL (Perkin Elmer).

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Budi E.H., Hoffman S., Gao S., Zhang Y.E, & Derynck R. (2019). Integration of TGF-β-induced Smad signaling in the insulin-induced transcriptional response in endothelial cells. Scientific Reports, 9, 16992.

Publication 2019

Bradford assays phospho-Akt (p-AktS473) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western Lighting Plus ECL

Antibodies Antibody Assays Bovine serum albumin Cell Glyceraldehyde 3 phosphate dehydrogenase Membrane Monoclonal antibodies Nitrocellulose Polyacrylamide gel Proteins Rabbit Sds page Smad2 Smad3

Corresponding Organization :

Other organizations : Broad Center, National Cancer Institute, Center for Cancer Research

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Variable analysis

independent variables

Cell lysis, protein extraction, and immunoblotting procedures

dependent variables

Protein concentrations
Protein expression levels of Smad2, phospho-Smad2, Smad3, Akt, and phospho-Akt
Protein expression levels of phospho-Smad3

control variables

Bovine serum albumin (BSA) used as a standard for protein quantification
SDS-PAGE on 4–12% gradient polyacrylamide gel
Transfer of proteins to a nitrocellulose membrane
Primary antibodies used for immunoblotting (Smad2, phospho-Smad2, Smad3, Akt, phospho-Akt, phospho-Smad3, GAPDH)
Western Lighting Plus ECL (Perkin Elmer) used for visualization of immunoreactive bands

controls

GAPDH used as a loading control

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