Western blot analysis
MP
LYSED, SECONDARY
Briefly, 5μg protein lysed from fibroblasts were loaded into each well of the gel. Proteins were separated using 4-12% Bis-Tris gel electrophoresis (Invitrogen), and then transferred to nitrocellulose membranes. Nonspecific binding was blocked by incubating the membranes using 5% nonfat milk diluted in 1x tris-buffered saline with 0.1% tween (TBST). Next, the membranes were treated with anti-CFD and anti-Col1a1 overnight at 4°C and then with a secondary antibody conjugated to HRP at room temperature. The chemiluminescence signal was identified and quantified by Image Lab (Bio-Rad ChemiDoc MP Imaging System) where the immune-positive bands were normalized with the band for the house-keeping gene, beta-actin (β-actin).