Journal: Cell Death and Differentiation

Article Title: IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3

doi: 10.1038/cdd.2014.10

Figure Lengend Snippet: Levels of protein for molecules that regulate apoptosis proximal to caspase-3. Equal amounts of whole-cell lysates from day 5 IL-2- and IL-15-cultured T cells were resolved by SDS-PAGE, and immunoblotted for (a) Bcl-2, Bcl-xL, and BimEL, (b) XIAP, cIAP-1, survivin, Smac/DIABLO, and cytochrome C, (c) RIPK1 and RIPK3, and (d) caspase-6 and caspase-7. Data are representative of at least two independent experiments

Article Snippet: Protein lysates were boiled for 5 min in loading buffer containing 2-ME and were separated by SDS-PAGE using 10 or 12.5% gels, transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad), and blocked using 4% milk in Tris-buffered saline plus 0.1% Tween-20 (American Bioanalytical, Natick, MA, USA) at room temperature for 1 h. Membranes were incubated at 4°C overnight in milk/Tris-buffered saline (TBS)-Tween containing mouse anti-caspase-8 monoclonal antibody (a kind gift of A Strasser, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia), rabbit anti-caspase-3 585 polyclonal antibody (the kind gift of Dr. Yuri Lazebnik, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA), mouse anti-caspase-9 monoclonal antibody (5B4) (Stressgen Assay Designs, Ann Arbor, MI, USA), rabbit anti-XIAP polyclonal antibody (Lifespan Biosciences, Seattle, WA, USA), rabbit anti-cIAP polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-survivin monoclonal antibody (Cell Signaling Technology), rabbit anti-Smac/DIABLO polyclonal antibody (BioVision, Milpitas, CA, USA), mouse anti-CytC monoclonal antibody (BD, San Jose, CA, USA), rabbit anti-hexokinase II monoclonal antibody (Cell Signaling Technology), or mouse anti-actin monoclonal antibody (Sigma-Aldrich), rabbit anti-caspase-6 (Cell Signaling), rabbit anti-caspase-7 (Cell Signaling), mouse anti-RIPK1 (BD Transduction Laboratories), rabbit anti-RIPK3 (ProSci Incorporated, Poway, CA, USA), rat anti-Bim (Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-Bcl-xL (Cell Signaling), or mouse anti-Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Cell Culture, SDS Page