Quantity one imaging analysis

The samples from cells or tissues were lysed and quantified. Twenty micrograms of protein from murine lung tissue or MH-S cells were mixed with an equal volume of 2×SDS sample buffer, boiled for 5 min and then separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes (Santa Cruz Biotechnology). Membranes were incubated with primary antibodies against FIP200, LC3, Beclin1, RAGE, ULK1, Histone, TLR4 and GAPDH (Santa Cruz Biotechnology). The antibodies against Atg13 and HMGB1 were from Cell Signaling Technology (Danvers, MA). Western blotting rabbit polyclonal antibodies, mouse polyclonal antibodies and goat polyclonal antibodies were obtained from Santa Cruz Biotechnology. Signals were visualized using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). The protein signal was quantified by scanning densitometry using Quantity one imaging analysis (Bio-Rad).

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).