c-KIT was enriched from whole cell lysates Emricasan mw by overnight incubation at 4°C with 1 μg mAb against c-KIT (104D2, Santa Cruz Biotechnology, Santa Cruz, CA), followed by immunoprecipitation with 50 μl Protein A Sepharose for 1 hr at room temperature, and three washes in buffer A. Proteins were eluted by boiling in NuPAGE LDS Sample buffer (Invitrogen),

separated by SDS-PAGE, and analyzed by Western blot using either c-KIT (104D2) or phosphorylated Tyr (PY20, Santa Cruz Biotechnology, CA) primary https://www.selleckchem.com/products/ly2090314.html antibodies at 1:1,000 dilution. Blots were developed using rabbit anti-mouse antibody coupled to HRP at 1:10,000 dilution and the ECL detection system (Amersham/GE Healthcare, Piscataway, NJ). Densitometry of individual bands was quantified using the ChemiDoc XRS system (Bio-Rad, Hercules, CA). The 60 kDa fraction of IgG was used as an internal loading control, and the percentage of phosphorylated c-KIT was calculated based on the normalized data for both total and tyrosine phosphorylated c-KIT. RelA/p65 activation assays THP-1 cells were incubated in media, with or without 1 μM OSI-930, for 5 h and then infected with Y. enterocolitica for 45 min at MOI 40. Cells were pelleted

and Androgen Receptor Antagonist purchase incubated in hypotonic lysis buffer NB (10 mM Tris, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5% NP-40, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM PMSF) for 15 min on ice. Cell nuclei were purified by centrifugation on 30% sucrose in NB buffer at 800 g for 10 min and resuspended in PBS/3.7% formaldehyde. Fixed cell nuclei were blocked in PBS/10% goat serum/1% BSA/0.1% Triton for 1h, incubated with 1:300 dilution of mouse anti-phospho-NFκB p65 (A-8, Santa Cruz Biotechnology) for 3 h, followed

by 1 h incubation in 1:500 dilution of goat anti-mouse IgG conjugated to Bupivacaine FITC (Abcam, Cambridge, MA), all at room temperature. After five washes in blocking buffer, the nuclei population was analyzed on a FACS CaliburII (Becton Dickinson, Franklin Lakes, NJ) using a blue laser (488 nm) and 530/30 emission channel with CellQuest Pro software. Flow cytometry analysis of c-KIT levels on cell membranes Formaldehyde (3.7%)-fixed NHDCs were rinsed with PBS containing 50 mM NH4Cl for 15 min. Cells were blocked with pre-immune heterologous serum (1:10 diluted in PBS) for 30 min, washed with PBS and incubated with primary phycoerythrin (PE)-conjugated c-KIT (Ab81, sc-13508PE, Santa Cruz Biotech, CA) for 4 h. The cell populations were acquired using a BD FACS CaliburII instrument with the blue laser (488 nm) and 585/42 emission channel and were analyzed using BD CellQuest Pro software. Statistical analysis Paired two-tailed Student’s t-test was used to calculate p-values, where ≤0.05 was considered statistically significant.