Examining Fibroblast Responses to Injury

Tissue samples of the posterior joint capsule collected at 0, 1, 3, 7, and 14 days following injury or primary joint capsule fibroblasts treated with various drugs (MIF, TGF-β1, siRNA or inhibitors) were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with 1 mM PMSF (Beyotime). Protein concentration in the supernatant was quantified using the Protein Assay Kit II (Bio-Rad, Inc., California, USA) to ensure equal loading. Western blots were performed as described previously 21 (link). The relative intensities of protein bands were normalized to those of β-actin. The following antibodies were used: β-actin (ProteinTech, Wuhan, China, 1:5000), MIF (Abcam, 1:1000), TGF-β1 (Abcam, 1:1000), α-smooth muscle actin (α-SMA, Abcam, 1:1000), Collagen I (Abcam, 1:1000), CD74 (Santa Cruz, CA, 1:100), p-ERK/ERK (Cell Signaling Technology, Danvers, MA, 1:1000), p-P38/P38 (Cell Signaling Technology, 1:1000), p-JNK/JNK (Cell Signaling Technology, 1:1000). Secondary antibodies included Goat anti-Rabbit IgG (H+L) (DyLight 800 4X PEG) (Invitrogen, 1:20000) and Goat anti-Mouse IgG (H+L) (DyLight 680) (Invitrogen, 1:20000). The fluorescent signals were determined with an Odyssey imaging system (Li-Cor, Lincoln, NE, USA).