Phospho sek

Cells were treated with 30 μΜ of 3-BDB for 1 h and then with 50 μg/mL PM_2.5 for 24 h, and mice skin tissues were treated with 3-BDB and PM_2.5 according to the above animal experiment method. Protein lysis buffers from the cells and mouse skin were loaded into a separating gel containing SDS-PAGE electrophoresis buffer. The target proteins were transferred onto membranes and shaken with primary and secondary antibodies sequentially. Finally, protein bands were obtained using the Amersham enhanced chemiluminescence, plus a Western blotting detection system (GE Healthcare, Buckinghamshire, UK). The primary antibodies used were as follows: actin (Sigma-Aldrich Co., Ltd.), c-Jun N-terminal kinase (JNK), p38 (Genetex Inc., Irvine, CA, USA), phospho-H2A.X, phospho-p53, caspase-9, caspase-3, mitogen-activated protein kinase kinases (MEK)1/2, phospho-MEK, phospho-extracellular regulated kinase (ERK), stress-activated ERK kinase (SEK)1, phospho-SEK, phospho-JNK, phospho-p38, c-Fos, c-Jun, phospho-c-Jun (Cell Signaling Technology, Danvers, MA, USA), B-cell lymphoma protein (Bcl)-2, Bcl-2 associated X (Bax), ERK2 (Santa Cruz Biotechnology, Dallas, TX, USA), IL-1β, matrix metalloproteinase (MMP)-2, MMP-9 (Abcam, Cambridge, MA, USA), p53, IL-6 (Invitrogen), MMP-1 (Cusabio, Houston, TX, USA).

Muscle tissues and myotubes were homogenized in lysis buffer containing 20 mM HEPES (pH 7.2), 150 mM NaCl, 0.5% Triton X-100, 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), and 5 mg/ml aprotinin (Sigma-Aldrich). The lysates were centrifuged at 15,000×g for 20 min at 4 °C, and the supernatants were subjected to SDS-PAGE followed by immunoblot analysis. Antibodies used are as follows: 4EBP1 (#9452), ATG5 (#12994), ATG7 (#8558), ATG12 (#4180), ATG16L (#8089), beclin1 (#3495), eIF2α (#9722), IRE1α (#3294), mTOR (#2983), p65 (#6956), PERK (#3192), S6K (#9202), SEK (#9152), SQSTM1 (#8025) phospho-4EBP1 (#9459), phospho-AKT S473 (#9271), phospho-GSK-3β (#9327), phosphor-mTOR (#5536), phospho-eIF2α (#9721), and phospho-PERK (#3179), phospho-JNK (#9251), phospho-SEK (#9151), phospho-S6K (#9206) from Cell Signaling Technologies; phospho-IRE1α (ab48187) from Abcam; and HA (sc-805), AKT (sc-1618), ATF6 (sc-22799), MYH (B-5, sc-376157), GSK-3β (sc-7291), and FABP3 (sc-58274) from Santa Cruz Biotechnology; and JNK (51-1570) from BD Bioscience; Laminin (L9393) from Sigma-Aldrich. The anti-GAPDH antibody was developed in our laboratory. Data were collected using Automatic X-ray Film Processor (JP-33, JPI America) or iBright FL1500 (Invitrogen).

Protein lysates (30 μg per lane) were electrophoresed on 12% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes, which were incubated with the primary antibodies and subsequently with HRP-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Thereafter, the membranes were developed using a western blotting detection kit (GE Healthcare Life Sciences, Buckinghamshire, Little Chalfont, UK) for detection of the protein bands and then exposed to X-ray film. The primary antibodies used in this study were as follows: MMP-1 (Cusabio Technology LLC., Houston, TX, USA), MMP-2 (Abcam, Cambridge, UK), MMP-9 (Abcam, Cambridge, UK), phospho-c-Jun (Cell Signaling Technology, Danvers, MA, USA), c-Fos (Cell Signaling Technology, Danvers, MA, USA), phospho-SEK (Cell Signaling Technology, Danvers, MA, USA), phospho-MEK (Cell signaling Technology, Danvers, MA, USA), phospho-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-JNK (Cell Signaling Technology, Danvers, MA, USA), and Actin (Sigma-Aldrich Chemical Company, St. Louis, MO, USA).