αnfatc1

ChIP assay was performed according to the manufacturer’s guidance (Invitrogen MAGnify ChIP system). Briefly, isolated CD3+ T cells were activated with αCD3/αCD28-conjugated beads for 24 hr and fixed with 2% formaldehyde. Sonicated DNA was immunoprecipiated with αSmad3 (Cell Signaling Technology), and αNFATc1 (Santa Cruz Biotechnology). The immunoprecipated chromatin was analyzed on Roche Light Cycler 480 by SYBR green using the following primers for PD-1 promoter. Pdcd-1: Forward 5′-CCTCACATCTCTGAGACCCG-3′; Reverse 5′-CCGAAGCGAGGCTAGAAACC-3′ Gapdh: 5′-TACTAGCGGTTTTACGGGCG-3′ 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′

Antibodies were obtained from the following sources: α-myc (Cell Signaling, Inc., Beverly, MA. Catalog number: 2272), α-hemagglutinin (HA) (Roche Applied Science, Indianapolis, IN. Catalog number: 11867423001), α-NFATc1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Catalog number: sc-7294), α-Mitf (C5) (Calbiochem, San Diego, CA. Catalog number: MAB3747-I and a gift from Dr. David E. Fisher at Massachusetts General Hospital, Boston, MA) and α-tubulin (Sigma-Aldrich, St. Louis, MO. Catalog number: T9026-100UL). Cyclosporine A (CsA), polyinosinic-polycytidylic acid and SigmaFAST protease inhibitor were purchased from Sigma-Aldrich. Recombinant murine M-CSF and RANKL were purchased from PeproTech (Rocky Hill, NJ). All primers and probes were purchased from Integrated DNA technologies (Coralville, IA).