Lama1

The total protein was extracted from the cells using the M-PER mammalian protein extraction reagent (Pierce, IL, USA) or from rat joint synovial tissues using the T-PER tissue protein extraction reagent (Pierce). Equal amounts of protein (25 μg per lane), estimated by a bicinchoninic acid (BCA) protein assay kit (Pierce), were loaded onto (11%) SDS-PAGE gels and transferred onto nitrocellulose membranes. The blots were probed with a monoclonal antibody against rat Smad4 (1:300), TGF-β (1:800), α-SMA (1:600), collagen I (1:800), collagen III (1:600), Lama1 (1:800), Timp1 (1:800) and beta actin (1:1200) (Santa Cruz, USA), followed by the secondary HRP-conjugated anti-mouse/rabbit antibody (Santa Cruz, USA). After washing, the bands were detected by chemiluminescence and imaged with X-ray films. beta actin was used as an endogenous reference for normalization.

Cellular proteins were extracted using radioimmunoprecipitation assay lysis buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer, the proteins were blocked with milk and probed with primary antibodies against the following: LAMA1 (Santa Cruz, CA, USA; sc-74418), MMP2 (Santa Cruz Biotechnology; sc-13594), MMP7 (Santa Cruz Biotechnology; sc-515703), MMP9 (Santa Cruz Biotechnology; sc-393859), GAPDH (ABclonal, Woburn, MA, USA; AC002), N-cadherin (Cell Signaling Technology, Danvers, MA, USA; 13116S), E-cadherin (ABclonal; A3044), Snail (Cell Signaling Technology, 3879S), Slug (Abcam, Cambridge, MA, USA; ab51772), and vimentin (Abcam; ab92547) at 4 °C overnight. Then, the membranes were incubated with secondary antibodies and exposed using the LI-COR Odyssey Imaging System (Lincoln, NE, USA).