Pstat 4

Recombinant GM-CSF, IL-4, and naïve CD4 T cell enrichment kit, Abs to IL-12p40/70, TNF-α, IL-6, IFN-γ, IL-17, PE-Cy7-CD11c, FITC-CD80, PE-CD86, efluor-MHCII, APC-CD40, STAT-1, pSTAT-1, STAT-4, pSTAT-4, JNK, and pJNK were purchased from BD Biosciences (San Diego, CA, USA). Biotinylated anti-CD83 and APC-conjugated CCR7 Abs were purchased from eBioscience (San Diego, CA, USA). Abs against actin were from Sigma (St. Louis, MO, USA), SOCS-3 from Abcam (Cambridge, MA, USA) and anti-pERK and ERK Abs from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The endotoxin removal column was purchased from Thermo Scientific (Rockford, IL, USA).

BM neutrophils from WT, STAT4^fl/flLysM^cre, and STAT4^fl/flS100A8^cre mice were isolated via negative selection kit (STEMCELL Technologies, 19762A) and stimulated with 40 ng/mL IL-12 (Peprotech, 210-12) in RPMI with 10% FBS for the indicated time points. Following stimulation, cells were washed and lysed with lysis buffer (Thermo Fisher Scientific, 78501) contacting protease inhibitor (Thermo Fisher Scientific, 78429) and phosphatase inhibitor cocktails (Cell Signaling Technology, 5870S). Protein content was determined by BCA method (Thermo Fisher Scientific), and 20 μg of protein lysate was resolved on a 4%–20% polyacrylamide gradient gel, followed by transfer to PVDF membrane via semidry transfer. Membranes were blocked at room temperature for 1 hour with TBS-based blocking buffer (LI-COR Biosciences 927-50000). The following primary Abs were incubated at 4°C overnight: STAT4 (Cell Signaling Technology C46B10), p-STAT4 (BD Biosciences 612739), JAK2 (Cell Signaling Technology 3230), p-JAK2 (Cell Signaling Technology, 3771), and GAPDH (Santa Cruz Biotechnology, 32233). Secondary Abs (926-68072 and 926-32211, Li-COR Biosciences) were incubated at room temperature for 1 hour. Images were acquired using the LI-COR Odyssey system. Band intensity was determined using Image Studio Lite software (LI-COR Biosciences).