T per solution

Individual embryos were solubilized in T-PER solution (Thermo, MA) containing cOmplete Protease Inhibitor Cocktail (Roche, Switzerland) on ice by gentle pipetting. After centrifugation at 12000 x g for 10 min at 4 °C, the supernatant was collected and the protein content was estimated using the Protein Assay Kit (Biorad, CA). Since total protein levels obtained from individual embryos were small, we loaded 16 μl of each sample in a 10% polyacrylamide gel for electrophoretic separation at 100 V for around 2 h. Then, proteins were transferred to a nitrocellulose membrane for 1 h at a constant current of 300 mA on ice. Membranes were blotted with antibodies raised against AR (rabbit polyclonal IgG 1:500; Santa Cruz Biotechnologies, TX) and Tubulin (TUBB; rabbit polyclonal IgG 160 ng/ml; Abcam, England), this latter as protein loading control. Antibody binding was detected with a second antibody raised in goat against rabbit IgG bound to peroxidase (1.8 μg/ml; Sigma, MO), revealed by chemiluminescence and documented using a G:Box Chemi XRQ system (Syngene, England). Band intensity was measured with the ImageJ 1.45 software. The intensity of each band was expressed as percentage of the average intensity of WT samples in each gel.

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)).

Half brain hemispheres, that were stored at − 80°C, were crushed on dry ice using mortar and pestle, then homogenized in T-PER solution (Thermo Scientific, Waltham, MA) and phosphatase and protease inhibitor mixtures (Thermo Scientific, MA and Roche, CA) and processed as previously described (Marsh et al., 2016 (link)). Quantitative biochemical analysis of human Aβ was conducted using commercially available electro-chemiluminescent multiplex assay system [Meso Scale Discovery (MSD)]. Human Aβ duplex (6E10 capture antibody) was used for simultaneous measurement of Aβ40 and Aβ42 in both soluble and insoluble protein fractions (Marsh et al., 2016 (link)).