Analysis was performed with IDEAS software (Amnis). Jurkat cells were labeled with DDAO Selleckchem RAD001 (Life Technologies) according to the manufacturer’s instruction, treated with 2.5 μg/mL Cycloheximide (Sigma-Aldrich) for 2 h, and added to CpG-activated (6 h) or resting CAL-1-NAB2, CAL-1-NAB2E51K, or CAL-1-EV in a ratio 25:1. For TRAIL blocking, 10 μg/mL anti-TRAIL (2E5; Enzo Life Sciences) was added to CAL1 cells 30 min prior to coculture with Jurkat

cells. After 20 h, apoptosis was measured with AnnexinV-PE staining (BD Biosciences) or with CaspGLOW Red Active Caspase-3 Staining Kit (BioVision) according to the manufacturers’ protocols. Total RNA was isolated with TRIZOL (Invitrogen). cDNA was generated with SuperScript RT II (Invitrogen) using Random Primers (Promega). Real-time RT-PCR was performed with ABsolute QPCR SYBR Green mix (Abgene) or SyBR Green Master Mix (Applied Biosystems) using the CFX96 (Bio-Rad) or Step One Plus (Applied Biosystems). SCH727965 The following primers were used for analysis: TRAIL (5′-ATGGCTATGATGGAGGTCCAG-3′;

5′-TTGTCCTGCATCTGCTTCAGC-3′), NAB2 (5′-CCCGAGAGAGCACCTACTTG-3′; 5′-GGGTGACTCTGTTCTCCAACC-3′), CD40 (5′-CGGCTTCTTCTCCAATGTGT-3′; 5′-ACCAAGAGGATGGCAAACAG-3′), IFN-β (5′-GAGCTACAACTTGCTTGGATTCC-3′; 5′- CAAGCCTCCCATTCAATTGC-3′), MXA (5′-TCCAGCCACCATTCCAAG-3′; 5′-CAACAAGTTAAATGGTATCACAGAGC-3′). 18s (5′-AGACAACAAGCTCCGTGAAGA-3′; 5′-CAGAAGTGACGCAGCCCTCTA-3′) was used as reference gene. The relative mRNA expression was calculated with the comparative CT (DDCT) method. Cell pellets were resuspended in 5× sample buffer or NP-40 lysis buffer containing protease inhibitors, and denaturated at 95°C. For NAB2 detection, cells were sonicated for 20 s prior to denaturation. SDS gels were transferred to nitrocellulose (Amersham Biosciences) or PVDF (Invitrogen) membranes, blocked with 5% nonfat milk or 4% BSA. Membranes were incubated with anti-NAB2 (1C4; Santa Cruz Biotechnologies), or anti-Actin (I-19; Santa Cruz Biotechnologies),

anti-Akt, anti-phospo-Akt, p38MAPK, anti-phospo-p38MAPK (Cell Signaling Technology), selleck inhibitor anti-NF-kB p65 (Santa Cruz Biotechnologies), anti-phospo-NF-kB p65 (Cell Signaling Technology), or anti-RhoGDI (BD Transduction Laboratories). Protein expression was revealed with HRP-conjugated secondary antibodies and assessed with ECL Plus Western Blot Detection Reagents (Amersham Biosciences or Thermo Scientific). TNF-α and IL-6 expression was measured in supernatants with the Cytometric Bead Array, according to the manufacturer’s protocol (CBA, Human Inflammation Kit, BD Biosciences). Data are represented as mean ± standard deviation (SD), and evaluated using a two-tailed, paired Student’s t-test (Geo MFI expression data), or a two-tailed, unpaired Student’s t-test (RT-PCR data and Apoptosis assay) unless stated otherwise. A probability value of p < 0.05 was considered statistically significant. We thank Dr. T.