Anti rabbit or anti mouse peroxidase conjugated secondary antibodies

Cell lysates were centrifuged at 12,000 rpm for 7 min at 4 °C. Protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA), and 10–15 μg of protein was loaded per lane onto 10% SDS–polyacrylamide gels, and the separated proteins were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% bovine serum albumin (BSA) with 0.1% Tween-20 and incubated with the appropriate primary antibodies overnight at 4 °C ((fibronectin: BD Pharmigen), (CD31, VE-cadherin, β-actin, total and phospho-cofilin, total and phospho-cell division cycle (cdc) 42, Rho A, total and phospho-smad2/3: Cell Signaling, Danvers, MA, USA), (total and phospho-NF-κB p65, and total and phospho-inhibitor of NF (I)κBα: Santa Cruz Biotechnology, Dallas, TX, USA); (α-smooth muscle actin (SMA) and TGF-βR1: Abcam, Cambridge, UK)). The bands were later incubated with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific). The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Advansta, Menlo Park, CA, USA) and quantified using the ImageJ(1.52v, NIH)software.

Proteins were extracted by centrifugation of the cell lysates at 4 °C for 7 min at 12,000 rpm. Protein concentration was determined using the Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (10–15 μg) were separated using 10% SDS-PAGE, and the separated proteins were transferred onto nitrocellulose membranes. Then, the membranes were blocked with 5% bovine serum albumin (BSA) with 0.1% Tween-20 at room temperature for 1 h, followed by incubation with appropriate primary antibodies (see Section 2.1) at 4 °C overnight. After washing with TBS containing 0.1% Tween-20, the membranes were incubated with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific). The bands were visualized using an enhanced chemiluminescence kit (Advansta, Menlo Park, CA, USA) and ImageJ (ver. 1.52v, NIH) software was used for quantification.