Anti phospho pkc

All cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Triton X-100 with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific). Primary antibodies used for western blotting include: anti-phospho-MBP (EMD Millipore, 05-429), anti-MBP (LifeSpan BioSciences, LS-C312288/59980), anti-flag-M2 (Sigma-Aldrich, #F1804), anti-HUNK (Invitrogen PA5-28765), anti-p27 (Santa Cruz, sc-528), anti-phospho-PKC-substrates (Cell Signaling, #2261), anti-phospho-PKC (Santa Cruz, sc-271920), anti-PKC (Santa Cruz, sc-17769), and anti-β-tubulin (Santa Cruz, sc-55529). Imaging was performed on the Protein Simple FluorChem-R imaging system.

Male Sprague–Dawley rats, weighing 180–220 g each, were obtained from the Animal Center of the Fourth Military Medical University. The study was approved by the Ethics Review board of the Fourth Military Medical University. All animal experiments were performed in accordance with the National Institute of Health’s guidelines for the care and use of laboratory animals (Publication No.85-23, revised 1985).
The formula seawater was prepared according to the composition of water from the East China Sea provided by Chinese Ocean Bureau: osmolality 1300 mmol/L, pH 8.2, specific gravity 1.05, NaCl 26.518 g/L, MgSO₄ 3.305 g/L, MgCl₂ 2.447 g/L, CaCl₂ 1.141 g/L, KCl 0.725 g/L, NaHCO₃ 0.202 g/L, and NaBr 0.083 g/L. The artificial seawater was confirmed to be sterile before endotracheal instillation. Anti-p-Ser368 of Cx43, anti-Cx43, anti-phospho-PKC, anti-PKC, and anti-β-actin antibodies for Western blot were purchased from Santa Cruz Biotechnology (Cell Signaling Inc). Staurosporine (STS, inhibitor of PKC) and phorbol myristate acetate (PMA, activator of PKC) were purchased from Beyotime (Nantong, China).