Whole cell extracts were prepared using RIPA buffer, resolved on SDS-PAGE gels, and transferred to acetate cellulose membranes. Primary antibodies used were anti-TAGLN (1:1000, Abcam plc.), anti-MYH11 (1:500, Abcam plc.), anti-ACTA2 (1:200, Sigma Corp.), anti-CNN1 (1:500, Sigma Corp.), anti-pSMAD2 (1:500, Cell Signaling Technology Inc.), anti-pS6 (1:500, Cell Signaling Technology Inc.), anti-GAPDH (1:2000, Santa Cruz Inc.). Secondary antibodies used were IRDye680RD Donkey anti-Mouse, IRDye680LT Donkey anti-Rabbit, IRDye800CW Donkey anti-Mouse, IRDye800CW Donkey anti-Rabbit (All secondary antibodies were from Licor Inc.). Licor western blot detection system was used for the dual-color imaging. ImageJ was used for the quantification of bands.
For the ELISA assay of the secreted TGF-β1, fresh medium (DMEM/F12, N2, Pen/Strep, all from Life technologies Corp.) without serum was applied to the cells and harvested after 24 h. ELISA assay was carried out using the Human TGF-beta 1 Quantikine ELISA Kit from R&D systems following the manufacture's guidelines.
For the ELISA assay of the secreted TGF-β1, fresh medium (DMEM/F12, N2, Pen/Strep, all from Life technologies Corp.) without serum was applied to the cells and harvested after 24 h. ELISA assay was carried out using the Human TGF-beta 1 Quantikine ELISA Kit from R&D systems following the manufacture's guidelines.