Cell lysis kit

Samples of 50 mg were weighed, and the Cell lysis kit (Bio-Rad, Hercules, CA) was then employed as a means of extracting protein on ice with fresh protease and phosphatase inhibitors, as well as freshly added Phenylmethylsulfonyl fluoride. Following lysis and centrifugation, equivalent quantities of protein were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto nitrocellulose membranes. Blocking buffer (Li-cor, Lincoln, NE) was used to block these membranes that were then stained using primary anti-IL-12p40 (1:500, Abcam), anti-MIP-1β (1:500, Abcam), anti-PDGF-BB (1:500, Abcam), anti-IL-1Ra (1:500, Abcam,), or anti-b-actin (1:2000, Santa Cruz Biotechnology, Dallas, TX) antibody for 12 hours at 4°C. A secondary antibody (Li-cor) diluted 1:10,000 was then used, followed by color detection via infrared laser imaging system (Odyssey, Li-cor).

Spleen, lung, liver, and tumor were excised 1 day after treatment and homogenized in the Cell Lysis Kit (Bio-Rad Laboratories) with the FastPrep-24 5G Homogenizer. The samples were centrifuged for 10 min at 13,000g, and the supernatant was collected.

Superoxide production in the placenta and renal cortex were measured using the lucigenin technique, as previously described by our lab (Shields et al. 2018). Briefly, rat placentas and 1 g of renal cortexes from NP and NP+IL‐17 rats were snap frozen in liquid nitrogen immediately after collection and stored at −80°C until further processing. Tissue samples were homogenized using the Bio‐Rad Cell Lysis Kit (Bio‐Rad, Hercules, CA) according to the manufacturer's instructions. The tissue lysate was incubated with lucigenin at a concentration of 5 μmol/L. The samples were allowed to equilibrate for 15 min in the dark, and the luminescence was measured for 10 sec with a BioTek Plate Reader (BioTek, Winooski, VT). Luminescence was recorded as relative light units per minute (RLUs/min). An assay blank containing lucigenin with no homogenate was subtracted from the reading before transformation of the data. Each sample was read three times and the average was used for data transformation. The protein concentration was measured using a protein assay with BSA standards (Pierce, 169 Rockford, IL) and the data are expressed as RLU/min/mg protein.

The right lung middle lobe was homogenized with a cell lysis kit (Bio‐Rad, Hercules, CA). Total protein concentration was assessed via Lowry protein assay (Bio‐Rad). Lung homogenates were diluted at various concentrations: 1:50, 1:100, 1:200 in 1% BSA (Sigma Aldrich) for ELISA. CCL5, CXCL1, IL‐5, IL‐6, IL‐17A, IL‐25, IL‐33, and TNF‐α protein levels were measured using Biolegend or R&D Systems (Minneapolis, MN) ELISA kits according to the manufacturer's protocol. Protein levels were standardized to total lung protein.

Phosphorylated proteins (Phosph-ERK1/2 (T202/Y204, T185/Y187); No. 5016616, Phospho-Akt (S473); No. 5017459, Phospho-p38 MAPK (T189/Y182); No. 5017183, and Bio-Plex® Phosphoprotein Detection Reagent Kit; No. 310013270, all from Bio-Rad, Germany) were analyzed with the Bio-Plex 200 Array System with Bio-Plex™ Manager 4.1.1. software (Bio-Rad, Germany) according to the manufacturer's recommendations. Total protein lysates (10 μg soluble protein) were prepared with cell lysis kit from Bio-Rad, Germany. Protein lysates were incubated overnight in 96-well plates with fluorescent capturing beads. Then, plates were washed and incubated with biotinylated antibodies against the phosphorylated proteins and final added streptavidin-phycoerythrin solution. The measurement of phosphorylated proteins was performed with the relative mean fluorescence intensity calculated excluding the blank (approach without protein lysates) (n = 3).