Immunoblotting
Both lung tissue homogenate and plasma samples were immunoblotted. Using Bradford assay, sample volume containing 25 µg protein was estimated. Sample volume containing 25 µg protein was separated on 10% SDS-PAGE gel and then transferred onto nitrocellulose/PVDF membrane. 5% skim milk in PBS-0.1% Tween-20 (PBST) was used to block the membrane at 4 °C overnight. The membrane was then washed with PBST thrice (10 min each) followed by sequential incubation with given primary and secondary antibodies for 2 h at room temperature. Secondary antibody was removed and membrane was again washed thrice with PBST. The membrane was then developed using chemiluminescent peroxidase substrate (Cat. no. CPS1300, Sigma, USA). ImageJ (http://rsbweb.nih.gov/ij/) was used for densitometric analyses of the autoradiograms. 1D SDS-PAGE gels were silver-stained and served as loading controls for both lung tissue homogenate and plasma samples. The following primary antibodies were used for immunoblotting: CLIC5 (Cat. # PA5-41047; Thermo Fisher, USA); GAPDH (Cat. # 21612, SAB, USA); Trx (Cat. # 46892-2, Santa Cruz, UK); RXR (Cat. # 33481, SAB, USA); MDH1 (Cat. # bs3396R, Bioss, USA); HIF-1a (Cat. # NB100105, Novus Biologicals, USA); STAT3 (Cat. # ab76315; Abcam, USA); GPx3 (Cat. # NBP1-06398, Novus Biologicals, USA); Plasminogen (Cat. # ab154560, Abcam, USA); C3 (Cat. # WH0000718M1, Sigma, USA); C4b (Cat. # Ab66791, Abcam, USA); Ttr (Cat. # MAB10762, Merck, USA); Apo A1 (Cat. # sc-30089, Santa Cruz, UK) and Apo H (Cat. # bs-1570R, Bioss, USA).
ELISA
ELISA was performed as per manufacturers’ instructions for the proteins alpha-1-antitrypsin (Cat. no. CSB-E11719h, Cusabio, USA), cofilin-1 (Cat. no. CSB-EL005280HU, Cusabio, USA) and S100A8 (Cat. no. CSB-E11833h, Cusabio, USA) using human plasma.