Western Blot Analysis of ECM Proteins

Proteins from cell lysates were separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk for 2 h at RT, and then incubated with anti-elastin (1: 2000, ab23747, Abcam, Cambridge, UK), anti-SMA (1:750, ab5694, Abcam, Cambridge, UK), anti-vimentin (1:2000, ab92547, Abcam, Cambridge, UK), E-cadherin (1: 2000, 3195, Cell signalling technology, USA) and anti-β-actin (1:10,000, ab8226, Abcam, Cambridge, UK) at 4 °C overnight. Blots were incubated with anti-rabbit or anti-mouse IgG HRP conjugated antibodies (R&D System, MN, USA) at RT for 2 h after TBS-Tween-20 wash (3 times, 10 min). Substrate (SuperSignal™ West Femto Maximum sensitivity substrate, Thermo Fisher Scientific, MA, USA) was added to the membrane and images of immunoblots were captured using a ChemiDoc MP System (Bio-Rad, Hercules, USA). Some blots were cut based on molecular weight and some blots were stripped only once for housekeeping protein. Densitometry analysis was performed relative to the housekeeping protein β-actin using ImageJ (NIH, Bethesda, USA) as previously described [23 (link), 24 ].