To obtain whole-cell lysates, MH-S cells or murine lung homogenates were homogenized in lysis buffer containing phosphatase inhibitor (1:1000) and Protease inhibitors (1:50, Roche, Indianapolis, IN). The samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentrations were determined by the BCA Protein Assay Kit (Bio-Rad) and then stored at −80 °C for immunoblotting analysis [37 ].
Whole-cell lysates were mixed with anti-FIP200 antibody (Cell Signaling Technology, Beverly, MA) or anti-HMGB1 antibody (Santa Cruz Biotechnology), respectively, which were coupled to agarose beads (Invitrogen). Nuclear Extracts were mixed with anti-acetylation on epsilon-amine groups of lysine residues antibody (Cell Signaling Technology) or anti-HMGB1 antibody (Santa Cruz Biotechnology) coupled with agarose beads. Immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose transfer membranes (Santa Cruz Biotechnology). Signals were visualized using an ECL kit (Santa Cruz Biotechnology). The protein signal was quantified by scanning densitometry using Quantity one imaging analysis (Bio-Rad).
Whole-cell lysates were mixed with anti-FIP200 antibody (Cell Signaling Technology, Beverly, MA) or anti-HMGB1 antibody (Santa Cruz Biotechnology), respectively, which were coupled to agarose beads (Invitrogen). Nuclear Extracts were mixed with anti-acetylation on epsilon-amine groups of lysine residues antibody (Cell Signaling Technology) or anti-HMGB1 antibody (Santa Cruz Biotechnology) coupled with agarose beads. Immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose transfer membranes (Santa Cruz Biotechnology). Signals were visualized using an ECL kit (Santa Cruz Biotechnology). The protein signal was quantified by scanning densitometry using Quantity one imaging analysis (Bio-Rad).