Anti total actin

Total protein extraction was performed on the endothelium and the tunica media of aortic tissue samples from MFS TAA and non-MFS TAA. Before proceeding, aortic adventitia was removed and the endothelium was scraped. In order to analyze the expression of End-Mt markers, filters were incubated with specific primary antibodies: mouse monoclonal anti-DNAJB6 (1:300, Thermofisher, Waltham, MA, USA), anti-cd31 (1:200, Dako, Santa Clara, CA, USA), anti-Vimentin (1:200, Santa Cruz Biotechnology), and anti-β-catenin (1:200, Santa Cruz Biotechnology). For fibrosis markers, filters were incubated with specific primary antibodies: mouse monoclonal anti-DNAJB6 (1:300, Thermofisher), anti-β catenin (1:200, Santa Cruz Biotechnology), and anti-ED-A FN (1:200, Santa Cruz Biotechnology). Normalization was performed using mouse polyclonal anti-α-tubulin (1:700, Sigma Aldrich) or rabbit polyclonal anti-total actin (1:500, Sigma Aldrich). Detection and quantification were carried out as reported [47 (link)]. For MFS TAA vs. non-MFS TAA aorta analysis (two for non-MFS and two for MFS; five samples for each pool), totaling ten MFS TAA patients and ten non-MFS TAA patients.

Reagent sources were as follows: MCT (Wako Pure Chemical Industries, Ltd., Osaka, Japan), l-NAME (Dojindo, Kumamoto, Japan), BAY11-7082 (Merck (Calbiochem), Darmstadt, Germany), PD98059 (Wako Pure Chemical Industries, Ltd.), SP600125 (Jena Bioscience, Jena, Germany) and SNP (Sigma Aldrich, St., Louis, MO, USA).
Antibody sources were as follows: anti-POSTN (1:100 dilution) (Proteintech, Rosemont, IL, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (1:1000 dilution) (GeneTex, Irvine, CA, USA), anti-iNOS (1:250 dilution for western blotting or 1:100 dilution for immunohistochemistry) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA or Bioss, Woburn, MA, USA), anti-total-actin (1:1000 dilution) (Sigma Aldrich), anti-phospho-VASP (1:500 dilution) (Abcam, Cambridge, UK), anti-phospho-ERK1/2 (1:1000 dilution), anti-phospho-JNK (1:250 dilution), anti-total-JNK (1:500 dilution), anti-phospho-NF-κB p65 (1:500 dilution) (Cell Signaling Technology, Madison, WI, USA), anti-total-ERK1/2 (1:100 or 1:200 dilution) (Santa Cruz Biotech, Santa Cruz, CA, USA or Bioss), anti-total-NF-κB p65 (1:500 dilution) (Santa Cruz Biotech), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (1:10,000 dilution) (Amersham Biosciences, Buckinghamshire, UK or Cell Signaling Technology).