Antibodies to actin

Fibroblasts at a density of 2×10⁵ cells per well were transfected with Ad-FST for 24 hours and lysed in RIPA lysis buffer (ATTO Corp., Tokyo, Japan) containing protease inhibitor (Pierce Mini Tablets, IL, USA). Meanwhile, the culture medium was collected to measure FST protein expression by Ad-FST. Lysates and culture medium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to polyvinylidene difluoride membranes (Merck Millipore Ltd., Darmstadt, Germany) using a transfer system (Mini Trans-Blot Cell and systems, Bio-Rad, Hercules, CA, USA). The blots were incubated with specific antibodies against MMP-1, MMP-13, TIMP-1, TIMP-2, TIMP-4, fibronectin, PAI-1, TRPV4, and α-SMA (Abcam, Cambridge, UK). After reacting with secondary antibodies, immunoreactive bands were visualized by a Western blot detection system (EzWest-Lumi Plus, ATTO Corp., Japan). To verify the amounts of loaded proteins, blots were stripped of bound antibodies and reprobed using antibodies to actin (Abcam).

Cells (2×10⁵ per well) were transfected with Ad-RLN for four hours, and lysed in RIPA lysis buffer (ATTO Corp., Tokyo, Japan) containing protease inhibitor (Pierce, Rockford, IL, USA). Meanwhile, the culture medium was analysed for RLN protein expression by Ad-RLN. Lysates and culture medium were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to polyvinylidenedifluoride (Pierce) membranes using a transfer system (Mini Trans-Blot® Cell and systems, Bio-Rad, Hercules, CA, USA). The blots were incubated with specific antibodies against MMP-1, MMP-13 (Young In Frontier Co., Ltd., Seoul, Korea), ERK1/2, phosphor-ERK1/2 Smad2, phosphor-Smad2 (Cell Signaling Technology, Danvers, MA, USA), beta galactosidase, α-SMA, fibronectin, TIMP 1, and TIMP 4 (Abcam®, Cambridge, UK). After reacting with secondary antibodies, immunoreactive bands were visualized by a western blot detection system (ATTO Corp.). To verify the amounts of loaded proteins, blots were stripped of bound antibodies and re-probed using antibodies to actin (Abcam®).