The human pro-inflammatory cytokines IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL_12p70, IL-13, and TNF-α were measured in 150 μL of neat lung lavage fluids by using electrochemiluminescent sulfo-tag labels as implemented in the multi-spot plates in Proinflammatory Human Cytokines Panel I V-plex kits (Meso Scale Discovery). Analyses were done using MESO QuickPlex SQ 120 and Discovery Workbench software (MSD). The expression of Endothelial-Monocyte Activating Polypeptide II (EMAP II) was assessed by Western blot analyses in mouse lung tissue homogenates. Briefly, lung tissues were homogenized in T-PER solution (Thermo Scientific) supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail; Sigma). The tissue lysates were analyzed by SDS-gel electrophoresis using AnykD TGX pre-cast gels (Bio-Rad). The following antibodies were used: rabbit polyclonal anti-EMAP II serum (lot D88) (86 (link)) or anti-AIMP1/EMAPII/SCYE1 (Bethyl laboratories # A304-896A-M), rabbit anti-Vinculin (Abcam #ab129002), along with HRP-conjugated goat anti-rabbit secondary antibodies. We imaged the membranes by incubation in Super-Signal West-Femto Substrate (Thermo Fisher) for five minutes, followed by a cumulative 3-min exposure time in Azure C300 imager (Azure Biosystems). Densitometry analyses were done using FIJI software (87 (link)).
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