Total protein

Serum was utilized to measure the levels of ALT, AST, and albumin by the ab234579, ab263883, and ab108789 kits purchased from Abcam, USA. In addition, the total proteins (BioRad, Hercules, CA, USA) and bilirubin (MBS730053, MyBioSource, San Diego, CA, USA) were measured in all experimental groups.

The collection and analysis of bronchoalveolar lavage (BAL) has been performed according to a previously described method (Hardy et al. 2009 (link); Nemmar et al. 2009 (link), 2011 (link), 2012b (link)). In brief, in separate animal groups (n = 8 per group), mice were sacrificed with an overdose of sodium pentobarbital after WPS or air exposure. The trachea was cannulated and lungs were lavaged three times with 0.7 mL (a total volume of 2.1 mL) of sterile NaCl 0.9% solution. The recovered fluid aliquots were pooled. No difference in the volume of collected fluid was observed between the different groups. BAL fluid was centrifuged (for 10 min at 1000 × g, 4°C). The supernatant was stored at −80°C until further analysis for total proteins (kit from Biorad, Munich, Germany), endothelin (kit from Cayman Chemical, Ann Arbor, MI), and lactate dehydrogenase (LDH) analysis by UV assay using a commercially available kit (Roche, Basel, Switzerland) in blood chemistry analyzer (Roche COBAS).

Cells were initially washed thrice with ice-cold PBS and then lysed in 100–400 μL lysis buffers. After lysis, the cellular debris was removed with the help of centrifuge, operated at 12,000 rpm and 4°C for 20 min. After the quantification of total protein (Bio-Rad, Mississauga, ON, Canada), the clarified protein lysates from each experimental condition was boiled for 5 min. Subsequently, the boiled lysates were subjected to electrophoresis in denaturing 8% SDS-polyacrylamide gel to identify AMPK and acetyl-CoA carboxylase (ACC) proteins. The separated proteins from the SDS-PAGE were then transferred to a nitrocellulose membrane and were blocked. The membranes were later probed with primary antibodies of interest and horseradish peroxidase-conjugated anti-rabbit IgG as secondary antibody. Finally, the position of proteins was visualized using the enhanced chemiluminescene (ECL) reagent.