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.