P38 mapk

Western blotting was performed to evaluate the protein expression levels of DEGs. The liver tissues were homogenized using a liquid nitrogen pre-cooled high-speed tissue homogenizer (Gering Scientific Instruments Ltd, Beijing, China) and lysed using 10 μM phenylmethanesulfonyl fluoride (PMSF, Beyotime Institute of Biotechnology, Jiangsu, China) and a 1% protease inhibitor cocktail (104 mM AEBSF, 80 μM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin, and 1.5 mM Pepstatin A; Sigma). The protein contents were analyzed with the BCA Protein Assay Kit (Vazyme Biotech Co., Ltd, Nanjing, China). Standard Western blotting was performed, and the blots were visualized via a chemiluminescent system using ImageQuant LAS 500 imaging instruments (GE Healthcare Life Sciences, Shanghai, China) and quantified using the Image J analyzer software. Antibodies against ERK 1/2, p-ERK1/2, p-NF-kB p65, MAPK p38, p-MAPK p38, JNK, and p-JNK were purchased from Cell Signaling Technology (Danver, MA, USA). Antibodies against TLR4, NLRP3, IL-1b, MYD88, and NF-kB p65 were obtained from Abcam (Cambridge, MA, USA).

Cells were lysed on ice for 30 min with radioimmunoprecipitation assay buffer (Geneall Biotechnology, Seoul, Korea) supplemented with 1× Protease Inhibitor Cocktail Kit 5 (Bio-Medical Science Co., Ltd., Seoul, Korea). Proteins were collected via centrifugation at 13,000 rpm and 4 °C for 30 min, and their concentrations were quantified using Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) and compared with a bovine serum albumin standard curve. An equal amount (30 µg) of protein sample from each treatment was separated using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were incubated with antibodies specific to the phosphorylation of NF-κB p-65, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) (Cell Signaling, Danvers, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Dallas, TX, USA) was used as the protein loading control. Protein signaling was detected using Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA), and the detected signals were imaged and quantified in terms of intensity using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

Proteins were extracted, submitted to PAGE and transferred to nitrocellulose membranes as described (59 (link)). Membranes were incubated with primary antibodies against β-actin (4967, Cell Signaling, Danvers, MA), NF-κB p65 (8242, Cell Signaling) p44/42 MAPK (ERK1/2; 9102, Cell Signaling), phospho-p44/42 (ERK1/2; 9101, Cell Signaling), p38 MAPK (9102, Cell Signaling), phosho-p38 MAPK (9211, Cell Signaling), SIRT2 (ab67299; Abcam, Cambridge, United Kingdom), SIRT3 (5490; Cell Signaling), α-tubulin (T5168; Sigma-Aldrich, Darmstadt, Germany), and HRP-coupled secondary antibodies (31430 and 31460; Invitrogen). Blots were revealed with the enhanced chemiluminescence Western blotting system (Advansta, San Jose, CA). Images were recorded with the Fusion Fx system (Viber Lourmat, Collégien, France). Full length blots are presented in Supplementary Figure 1.

Phorbol ester phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLF), C5a and isoluminol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The p38 MAPK inhibitor SB203580 was ordered from Selleck (Houston, TX, USA). Antibodies including Phosphor-Akt, Akt, Phosphor-p38 MAPK, p38 MAPK, MK2, and GAPDH were ordered from Cell Signaling Technology (Danvers, MA, USA). Anti-p47^phox antibody was purchased from Santa Cruz Biotechnology and anti-phospho-p47^phox (Ser329) antibody was generated by N.J. Compass Biotechnology (Nanjing, China). Other reagents were ordered from Sigma-Aldrich (St. Louis, MO, USA).

Reagents. DMSO (Sigma, D8401), SB203580 (Calbiocam, 559389), SU6656 (Sigma, S9692), and RGD peptides (Sigma, A8052).
Antibodies. Fibronectin (BD Biosciences, 610077), α-tubulin(Sigma, T5168), β-actin (Sigma, A5441), integrin β-1 (Cell Signaling Technology, 4706), integrin β-3(D7X3P) (Cell Signaling Technology, 13166), integrin β-4 (Santa Cruz Biotechnology, sc-514426), integrin β-5 (Santa Cruz Biotechnology, sc-5402), integrin α-5 (Cell Signaling Technology, 4705), integrin α-v (Cell Signaling Technology, 4711) ERK1 (Santa Cruz Biotechnology, sc-94), p-ERK(T202/Y204) (Cell Signaling Technology, 9101), AKT (Cell Signaling Technology, 9272), p-AKT(S473) (Cell Signaling Technology, 9271), SAPK/JNK (Cell Signaling Technology, 9258), p-SAPK/JNK(T183/Y185) (Cell Signaling Technology, 9251), Smad2/3 (Cell Signaling Technology, 3102), Src (Cell Signaling Technology, 2110), p-Src(Y416) (Cell Signaling Technology, 2101), p38MAPK (Cell Signaling Technology, 9211), p-p38MAPK (Cell Signaling Technology, 9212), MAPKAPK2 (Cell Signaling Technology, 3042), p-MAPKAPK2(Thr334) (Cell Signaling Technology, 3007), and), p-paxillin (Y118) (Cell Signaling Technology, 2541).