Sm α actin

Antibodies directed against the following proteins are listed in Supplementary Table 2: SRSF1, SM-α-actin, PCNA, p21, EGR1, GFP, Myc, p53, β-actin, Eif-5, KLF5, Bcl-xL.Bcl-xL Angiotensin II (Ang II), and PDGF-BB were from Sigma. Unless indicated otherwise, all other chemicals were from Sigma-Aldrich.

Immunohistochemistry assay was performed as described in our previous report (Wang et al., 2011 (link)). To detect HMGXB4 expression, LCA and RCA were harvested from WT mice post‐surgery 21 days and fixed in 4% paraformaldehyde, then embedded in paraffin. Paraffin embedded tissues were sectioned at a thickness of 8 μm and then stained with HMGXB4 (Sigma, HPA000725, 1:200) or ACTA2 (SM α‐actin; Sigma, A2547, 1:000) antibody. For detection of primary antibodies, we used avidin‐biotin method with diaminobenzidine substrate as chromogen (brown; Vector Laboratories; Cat. No. SK‐4100). Sections were lightly counterstained using hematoxylin to visualize cell nuclei (blue). Control sections were processed identically as experimental samples except no primary antibody was applied.

The total, cytoplasmic and nuclear proteins were isolated using protein extraction kit (Beyotime) according to the manufacturer’s instructions. After quantitation (BCA kit, Beyotime), protein was separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred onto PVDF membrane. Antibodies against β-actin, β-catenin, smooth muscle myosin heavy chain protein (SM-MHC), Notch1 (Santa Cruz, CA, USA), GAPDH, histone H3, phospho-p38, phospho-ERK1/2 (Cell Signaling Technology, MA, USA) and smooth muscle α-actin (SM α-actin) (Sigma) were used to probe with the interest blots. Finally, protein expression was detected with HRP-conjugated second antibody (Santa Cruz) and ECL (Pierce, IL, USA) luminescence method.

Tannic acid was from Sigma-Aldrich (catalog # 403040). TGF-β was from EMD Millipore (catalog # GF111). Pharmaceutical grade bleomycin was from Teva Pharmaceuticals. Antibodies for Western blotting against SM α-actin (catalog # A5228), β-actin (catalog # A5441), and α-tubulin (catalog # T6074) were from Sigma-Aldrich; against human collagen-1A1 was from Santa Cruz Biotechnology (catalog # sc-28657); against mouse collagen 1 was from EMD Millipore (catalog # 234167); against Smad2 (catalog # L1603) and phospho-Smad2 (catalog # 13804) from Cell Signaling Technology. The SBE4-Luc plasmid was kindly provided by Dr. Bert Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD). pRL-TK plasmid was from Promega.

Frozen vascular samples were pulverized by bead-homgenization (TissueLyser, Qiagen, Germantown, MD), suspended in the presence of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer (125 mmol/l Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 100 mmol/l PMSF, 10 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin A), and sonicated, as previously reported (Atkins et al. 2005 (link), 2007 (link); Park et al. 2005 (link)). Protein concentration was determined (Pierce BCA, Thermo, Rockford, IL) and lysates (50 μg) were run on 7.5 or 10% SDS-PAGE and immunoblotted with antibodies for GLUT4 (MW∼55 kD – Abcam, Cambridge, MA), pERM (MW∼90 kD), pMYPT (MW∼130 kD – Thr850), and COX-2 (MW∼72 kD – Santa Cruz Biotechnology, Santa Cruz, CA), and MYPT (MW∼130 kD – BD Biosciences, San Jose, CA). All blots were within the linear range and, with the exception of those for pMYPT (normalized to total MYPT), were normalized to SM α-actin (MW∼41 kD) or α-actinin (MW∼100 kD – Sigma, St Louis, MO) the expression of which we have found are unaffected by hypertension (Armoni et al. 2014 (link)). Immunoblots for GLUT4 exhibit multiple bands due to the highly glycosylated nature of this protein (Brosius et al. 1992 (link); Katz et al. 1995 (link); Atkins et al. 2001 (link), 2005 (link), 2007 (link); Park et al. 2005 (link)).