Anti p38

Isovitexin (CAS No. 38953-85-4, purity: 98.01%) was obtained from Mansite Biotechnology, Chengdu, China) was dissolved in 100% DMSO at a concentration of 100 mM as a stock solution and diluted test concentration with culture medium before each experiment. The concentration of final DMSO did not extend 0.1% throughout the trail. Ginkgolic acids were purchased from Jingzhu Medical Technology (Nanjing, China). The Annexin V-FITC/PI apoptosis kit and ELISA kits for murine (TNF-α, IFN-γ, IL-2, IL-17A) were purchased from MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China). SHP099, the SHP2 inhibitor, purchased from Selleckchem. Concanavalin A (Con A), MTT, Freund’s Adjuvant Complete (FAC) and Dexamethasone (Dex) were purchased from Sigma-Aldrich (St Louis, MO). Antibodies against phospho-STAT3 (Tyr705), phospho-AKT (Ser473), Anti-ERK, phospho-P38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK1/2 (Thr 202/Tyr 204), Caspase-3, Caspase-8, Cleaved Caspase-3 (Asp175), Cleaved Caspase-8 (Asp387), Cleaved PARP (Asp214) were purchased from Cell Signal Technology (Beverly, MA). Antibodies against phospho-SHP2 (Tyr 542) was purchased from Abcam (Cambridge, United Kingdom). Anti-β-actin, Anti-AKT, Anti-PARP, Anti-P38, Anti-JNK, Anti-AKT, Anti-STAT6 was purchased from Proteintech Group (Wuhan, China). Anti-STAT3, Anti-SHP2 was purchased from Santa cruz Biotechnology.

At each indicated time point, cells from each group were washed twice with ice-cold PBS supplemented with 1 mM sodium vanadate and lysed in modified radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris (pH 7.4), 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics Ltd., Taipei, Taiwan) and 1 mM sodium vanadate. The lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. The proteins were quantitated using the BCA protein assay. The protein expression levels were analyzed by Western blotting using antibodies against BMP-2 (catalog number: bs-1012R; Bioss, Beijing, China), anti-p38 (catalog number: 14064-1-AP; Proteintech, Rosemont, IL, USA), anti-p-p38 (catalog number: AP0526; ABclonal, Wuhan, China), anti-JNK (catalog number: ARG51218; Arigo, Hsinchu, Taiwan), anti-p-JNK (catalog number: ARG51807; Arigo, Hsinchu, Taiwan) and β-actin (catalog number: A5441), and immunoreactions were visualized using an enhanced chemiluminescence (ECL) system (Amersham, UK).

The PAs homogenate tissue and collected HPASMCs and HPAECs were dissolved in proteolytic buffer for 30 minutes. After 30 minutes, the lysed PAs homogenate tissue and cell proteins were centrifuged at 13 500 g for 15 minutes, and then the protein concentration was measured using a BCA kit. Thirty micrograms of cell lysate from each sample were used for SDS‐PAGE (Bio‐Rad Laboratories), and Western blotting were analysed according to the protocol as described previously.²³ The chosen antibodies included anti‐NDUFA4L2 (Catalogue number: 16480‐1‐AP; ProteinTech), anti‐HIF1α (Catalogue number: AF1009; Affinity), anti‐PCNA (Catalogue number: 10205‐2‐AP; ProteinTech), anti‐cyclin A (Catalogue number: 13295‐1‐AP; ProteinTech), anti‐cyclin E (Catalogue number: 11554‐1‐AP; ProteinTech), anti‐ERK (Catalogue number: 16443‐1‐AP; ProteinTech), anti‐p‐ERK (Catalogue number: AF1015; Affinity), anti‐5‐LO (Catalogue number: AF4699; Affinity), anti‐p‐5‐LO (Catalogue number: AF8359; Affinity), anti‐p38 (Catalogue number: 14064‐1‐AP; ProteinTech), anti‐p‐p38 (Catalogue number: 4511T; Cell Signaling), anti‐JNK (Catalogue number: 51151‐1‐AP; ProteinTech) and anti‐p‐JNK (Catalogue number: 4668S; Cell Signaling) et al. After extensive washes membranes with TBS‐T, the ECL luminescent solution is added for exposure and development.

Western blot was performed as previously described^{20 (link)}. Antibodies used in this study were PDLIM1 (Affinity, DF3003), α-SMA (Affinity, AF1032), COL1A1 (Cell Signaling Technology, #91144), CTCF (Millipore, 07-729), TNF-α (Affinity, AF7014), IL-6 (Affinity, DF6087), p65(Affinity, AF5006), anti-p-Smad2/3 (Abcam, ab254407), anti-Smad2/3 (Abcam, ab202445), anti-p-ERK (Abcam, ab201015), anti-ERK (Abcam, ab184699), anti-p-P38 (Proteintech, 28796-1-AP), anti-P38 (Proteintech, 66234-1-Ig), anti-p-JNK (Proteintech, 80024-1-RR), anti-JNK (Proteintech, 66210-1-Ig), GAPDH (Cell Signaling Technology, #2118). Western blot analysis was performed on at least three independent biological replicates.

The skin, subcutaneous connective, and muscle tissues of the ST36 area were collected and fixed, and paraffin sections were prepared. Slices were deparaffinized in xylene and rehydrated with ethanol. Antigen retrieval was performed by microwave treatment of slices in EDTA Antigen Retrieval Solution (pH 9.0). The cells were treated with PBS (pH 7.4) and 3% BSA (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Cat# A8020), followed by incubation with the primary antibody (anti-p38 [1:100, Proteintech] or anti-F-actin [1:400, Proteintech]) at 4 °C overnight. After washing, goat anti-mouse RedX secondary antibody (1:400) was added, and samples were incubated in the dark for 45 min at 37 °C. Cells were washed with PBS and counterstained with DAPI, followed by spontaneous fluorescence quenching reagent and the application of a coverslip [31 (link)]. PBS was incubated as a negative control to ensure the specific binding of antibodies to the target proteins in immunofluorescence (Figure S2). The slices were observed and photographed under a fluorescence microscope (OLYMPUS CX53 & DP70, OLYMPUS, Tokyo, Japan). Fluorescence quantification was performed using DP2-BSW application software (Version 2.2, OLYMPUS, Japan).

Proteins from cells were lysed with a RIPA lysis buffer containing 1 mM PMSF (Beyotime) referring to the manufacturer’s instruction. Then, the concentration was measured by BCA Protein Assay Kit (Beyotime). The proteins were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Sigma-Aldrich). The membranes were blocked in 6% skim milk for 2 h and then incubated with primary antibodies at 4 °C overnight and finally with the secondary antibody (1:6000; Bioworld, Nanjing, China) for 1 h. The following primary antibodies were used: anti-GAPDH (1:6000; Proteintech™, WuHan, China), anti-RUNX2 (1:1000; Cell Signaling Technology, MA, USA), anti-OPN (1:1000, Proteintech™), anti-ALP (1:2000; Proteintech™), anti-COLLAGEN type I (1:2000;Proteintech™), anti-SMAD2(1:1000; Cell Signaling Technology), anti-phosphorylation SMAD2 (1:1000; Beyotime), anti-SMAD3 (1:1000; Beyotime), anti-phosphorylation SMAD3(1:1000; Beyotime),anti-ERK1/2(1:2000; Proteintech™), anti-phosphorylation ERK1/2(1:1000; Beyotime), anti-JNK (1:3000; Proteintech™), anti-phosphorylation JNK (1:1000; Beyotime), anti-P38(1:500; Proteintech™), and anti-phosphorylation P38(1:1000; Beyotime). The visualization of the protein bands was used with enhanced chemiluminescence kit (Beyotime).