Protease inhibitor cocktail

Cells were washed once with PBS buffer, lysed in IP Lysis Buffer (20 mM Tris-HCl, pH 7.4, 0.5% Nonidet-P40, 150 mM NaCl, 1x protease inhibitor cocktail [11836170001; Roche], 1x phosphatase inhibitor cocktail [PHOSS-RO; Roche], and 1 μM veliparib [S1004; Selleck Chemicals]) and centrifuged at 20,000 × g for 20 min at 4°C. In all, 5% of the supernatants were saved as input. In total, 25 μl of the Protein G Dynabeads (10004D; Thermo Fisher Scientific) or Protein A Dynabeads (10001D; Thermo Fisher Scientific) were incubated with primary antibody for 2 h with rotation at room temperature. Antibodies used for immunoprecipitation include HA (sc-7392; Santa Cruz) and IRF3 (4302; CST). Protein concentration was determined by BCA assay (23225; Thermo Fisher Scientific), and equal amounts of protein were incubated with antibody-bound beads with rotation overnight at 4°C. Beads were washed three times with IP buffer. IP complex was eluted in 1x Western Blot Sample Buffer (EC887, 5x; National Diagnostics) and boiled at 95°C for 5 min. 5x Protein Loading Buffer from National Diagnostics contains 1.0 M Tris-HCl (pH 8.5), 8% (wt/vol) lithium dodecyl sulfate, 40% (vol/wt) glycerol, 2 mM EDTA, 0.5 M DTT, and tracking dye in distilled/deionized water. 1x western blot sample buffer was made from dilution of 5x protein loading buffer with IP lysis buffer.

Lungs were excised and collected in PBS containing a 2× cOmplete, EDTA-free protease inhibitor cocktail (Roche). Lungs were homogenized using a mixer mill. Supernatants of homogenates were collected, and cytokines in the samples were analyzed using the BD cytometric Th1/Th2/Th17 kit (BD Biosciences). The kit was used for the simultaneous detection of mouse interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor (TNF), interleukin-17A (IL-17A), and interleukin-10 (IL-10) in a single sample. The operations were formed according to the manufacturer’s instructions. Beads coated with seven specific capture antibodies were fixed. Subsequently, 50 µL of the mixed captured beads, 50 µL of the unknown lung supernatant or standard dilution, and 50 µL of phycoerythrin (PE) detection reagent were added to each assay tube and incubated for 2 hours at room temperature in the dark. The samples were washed once with 5 mL of wash buffer and centrifuged. The bead pellet was resuspended in 300 µL buffer. Samples were measured on the CytoFLEX S (Beckman Coulter) Flow Cytometer equipped with four laser lines (405 nm, 488 nm, 561 nm, and 633 nm) fitted with filters FITC (525/40), PE (585/42), ECD (600/20). Individual cytokine concentrations were indicated by their fluorescent intensities. Cytokine standards were serially diluted to facilitate the construction of calibration curves, necessary for determining the protein concentrations of test samples.