P p38 p38

All inhibitors were purchased from Selleck Chemicals (Houston, TX), prepared as 10 mM stock solutions in DMSO, and further diluted with culture medium before use. Annexin V-FITC/PI apoptosis detection kit and cell cycle detection kit were purchased from KeyGen BioTECH (Nanjing, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, and Hoechst 33342 were obtained from Solarbio (Beijing, China). Antibodies for Src/p-Src, Mek/p-Mek, Erk/p-Erk, PI3K/p-PI3K, PDK1/p-PDK1, AKT/p-AKT, mTOR/p-mTOR, PTEN/p-PTEN(S380/T382/383), JNK/p-JNK, c-Jun, p38/p-p38, JAK2/P-JAK2, Stat3/p-Stat3, Rb/p-Rb, CDK4, CDK6, Cyclin D1, CDK2, Cyclin E1, RAD51, Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-7 were all purchased from Cell Signaling Technology (Danvers, MA, USA). Tubulin and GAPDH primary antibodies were purchased from ZSGB-Biotechnology (Beijing, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse IgG were obtained from BBI life sciences (Shanghai, China).

The protein lysis buffer containing a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China) was used to harvested cells. The cell suspensions were centrifuged at 4°C for 30 min with a speed of 12,000 × g. The BCA Protein Assay (CWBIO, Beijing, China) was used to determine the protein concentration, and each lane was loaded with equal aliquots of the total protein (20 μg). The sample lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked in blocking sodium (Beyotime, Shanghai, China) for 1 h, and probed with the following antibodies at 4°C overnight: DSPP (1 : 1000; Santa Cruz Technology, Santa Cruz, CA), DMP1 (1 : 1000; Bioss, Beijing, China), p38, p-p38, and β-actin (1 : 10000; Cell Signaling Technology, Beverly, MA, USA). The membrane was incubated at room temperature for 1 h with horseradish peroxidase- (HRP-) conjugated antirabbit immunoglobulin. The protein expression was detected using a Western enhanced chemiluminescence blotting kit (ECL, SOLIBRO, Beijing, China).

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).

Western blot assay was performed according to previous protocols [11 (link)]. The protein expression of cleaved-caspase-3 (cleaved-cas-3) and cleaved-caspase-9 (cleaved-cas-9), phosphorylation-c-Jun N-terminal kinase (p-JNK) and JNK, phosphorylation-p38 the mitogen-activated protein kinase (p-p38), p38, GADPH was measured by western blot in N2a cells. First, N2a cells were lysed by RIPA buffer (Beyotime). Then, the proteins were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Beyotime) and polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Afterward, the primary antibodies were incubated with the blocked membrane for 12 h at 4°C. In order to eliminate the unwanted distractions, 1× tris-buffered saline tween-20 (TBST) was applied to wash away the excess residues. The secondary antibody was incubated with the complexes for 1 h at room temperature. Eventually, the interested protein expressions were measured by Electrochemiluminescence detection kit (Roche Diagnostics GmbH, Mannheim, Germany). The antibodies were cleaved-cas-3, cleaved-cas-9, p-JNK and JNK, p-p38, p38 (1:1,000; Cell Signaling, Danvers, MA, USA), GAPDH (1:500; Santa Cruz, Dallas, TX, USA) and HRP-conjugated secondary antibodies (1:5,000; Southern-Biotech, Birmingham, AL, USA).