Western Blot Analysis Protocol

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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Kraus L., Ma L., Yang Y., Nguyen F., Hoy R.C., Okuno T., Khan M, & Mohsin S. (2020). Cortical Bone Derived Stem Cells Modulate Cardiac Fibroblast Response via miR-18a in the Heart After Injury. Frontiers in Cell and Developmental Biology, 8, 494.

Publication 2020

Mini-PROTEAN TGX Gels MAB374 EP784Y ab8978 IRDye secondary antibodies

Acta2 Antibodies Assay Ctgf Gapdh Gels Light Mouse Rabbit Smad2 Vimentin Western blot analysis

Corresponding Organization : Temple University

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Variable analysis

independent variables

Primary antibodies against Acta2, GAPDH, CTGF, P-SMAD2, SMAD2, and Vimentin

dependent variables

Protein expression levels of Acta2, GAPDH, CTGF, P-SMAD2, SMAD2, and Vimentin

control variables

Sample concentrations were determined using the Bicinchoninic assay (BCA) according to manufacturer's protocol
Ran on a Mini-PROTEAN TGX Gels (Bio-rad)
Primary antibodies were incubated overnight at 4°C
Incubated with the appropriately conjugated light-sensitive IRDye secondary antibodies (1:5000, LiCOR) for 1 h at room temperature

controls

No positive or negative controls were explicitly mentioned in the protocol

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