Given our findings, it seems classical, as well as novel PKC isoenzymes, may be capable of regulating thymocyte apoptosis in the absence of PKCθ. The association of Nur77 and PKC further exemplifies the significance of how these molecules act in concert to mediate a crucial component of thymocyte development. Cante-Barret et al.28 have shown that PKC regulates Bim transcription during negative selection; thus, PKC can activate at least two apoptotic pathways converging at mitochondria. Further studies are necessary to more clearly elucidate their role in negative selection. The PKCα and -θ antibodies were provided by Cell Signaling and Santa Cruz, respectively.

Pritelivir order The anti-CD3 (clone 2C11) and anti-CD28 (clone PV-1)

antibodies were purchased from the University of California, San Francisco, Hybridoma Facility. All other antibodies and reagents have been described previously 20. Bcl-2 BH3 intracellular staining was done as described 20. The Nur77 Serine-354-Alanine (S354A) mutant in the pSG5 vector backbone was generously provided by Dr. Lester Lau (University of Chicago) through Dr. Philippa Melamed. Nur77 and the Nur77(S354A) mutant were cloned into the MSCV 2.2-ires-GFP retroviral vector, a gift from Dr. William Sha (Berkeley). The VSV-G and a gag-pol helper plasmid for retroviral transduction were from the Nolan laboratory (Stanford). Thymocytes were stimulated with PMA or 1 μM HK434 plus ionomycin or plate-bound anti-CD3 (10 μg/mL) anti-CD28 (2 μg/mL). One-hour pre-treatment with 1 μM Gö6976 or GF109203X or 10 μM SB 203580 Selleck PD98059 or U0126 or 50 μM LY294002 or 20 μM SB600125 was used where indicated. All animal-related experiments have been approved by the Berkeley Animal Use and Care Committee. Phoenix cells were transfected with MSCV, VSV-G and gag-pol helper plasmids by Lipofectamine

2000 (Invitrogen) according to the manufacturer’s protocol. Five hours after transfection, the media was changed to Opti-MEM supplemented with 10% FCS, penicillin/streptomycin and α-mercaptoethanol (16610D9 media). Two days after transfection, the viral supernatant was syringe filtered (0.45 μm), supplemented with 4 μg/mL polybrene and added to 2.5×106 16610D9 cells. The cells were spun at 2500 rpm for 1 h and cultured for 2 days, with fresh 16610D9 Orotidine 5′-phosphate decarboxylase media added 24 h after infection, before cell fractionation. Retrovirally transduced 16610D9 cells were stimulated with 2.5 ng PMA/0.5 μM ionomycin for 2 h. After washing 1.5×107 16610D9s with PBS, cells were resuspended in 200 μL Solution A (10 mM HEPES-KOH [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF, 1 mM DTT and 0.5–0.6% Nonidet P40). They were then incubated on ice for 10 min and spun down briefly. The nuclear pellet was washed three times with PBS and resuspended in 40 μL 16610D9s of Solution B (20 mM HEPES-KOH [pH 7.9], 400 mM NaCl 20% glycerol, 0.2 mM EDTA, 0.2 mM PMSF, 1 mM DTT and 0.5–0.6% Nonidet P40).