2.7. Detection of Proteins Expression (iNOS, COX-2, NF-κB, VEGF, and TGF-β) via Western Blot

HDF cell protein extracts were harvested and prepared by using RIPA buffer for western blot analyses. The concentration of protein was determined by using bicinchoninic acid (BCA) assay according to the manufacturer's protocol. An equal amount of cellular proteins (20 μg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis under reducing conditions (incubated with 5× laemmli buffer) for separation. The separated proteins in gel were then transferred to polyvinylidene difuoride (PVDF; GE Healthcare) membrane and was allowed to set for 1 h. The membrane underwent blocking for 1 h with the use of a blocking solution (5% of BSA in phosphate-buffered saline containing 1% Tween-20 (PBST)) at room temperature prior to incubation of specific primary antibodies such as VEGF, TGF-1β, iNOS, COX-2, NF-κB, or β-actin at 4°C overnight. The membrane was washed 5 times with PBST followed by incubation with respective anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase for 1 h. It was followed by washing 5 times with PBST for 10-min intervals.

The bands were visualized under chemiluminescence system (Chemi Doc, Bio Rad, USA), followed by Image J software analysis.