Journal: Molecular and Cellular Biology
Article Title: FAST Is a Survival Protein That Senses Mitochondrial Stress and Modulates TIA-1-Regulated Changes in Protein Expression
doi: 10.1128/MCB.24.24.10718-10732.2004
Figure Lengend Snippet: FAST inhibits Fas- and UV-induced apoptosis. (A) HeLa cells were mock transfected or transfected with vectors encoding HA-FAST or β-galactosidase, cultured in the absence (untreated) or presence of anti-Fas antibody (FAS) before being processed for immunofluorescence microscopy using anti-HA, anti-β-galactosidase, anti-active caspase 3, and Hoechst dye. Arrows point out cells undergoing Fas-induced caspase-dependent apoptosis. Arrowheads point out FAST transfectants in which caspase 3 is not activated. Size bar, 20 μm. (B) HeLa cells were mock transfected or transfected with either FAST or β-galactosidase (β-gal), cultured in the presence of anti-Fas antibody, and then processed for TUNEL analysis (red) and immunofluorescence (anti-HA, green; anti-active caspase 3, blue) or Hoechst staining. Arrows point out untransfected cells or β-galactosidase-transfected cells. Arrowheads point out FAST transfectants. (C) The mean percentages (±standard errors; n = 3) of transfected cells (vector control, HA-FAST, HA-FASTN, or HA-FASTC; revealed using anti-HA or anti-β-galactosidase) cultured under the indicated conditions that exhibit active caspase 3 are presented as a bar graph. Calculated P values for selected comparisons are shown.
Article Snippet: Antibodies obtained from commercial sources include antihemagglutinin (anti-HA) (murine monoclonal antibody clone 16B12, immunoglobulin G1; Berkeley Antibody Co.), anti-FLAG (murine monoclonal antibody clone; Sigma Chemical Co., St. Louis, Mo.), anti-BCL-XL (rabbit polyclonal and mouse monoclonal; Santa Cruz Biotechnology, Inc.), anti-Myc (rabbit polyclonal; Santa Cruz Biotechnology, Inc.), anti-active caspase 3 (rabbit polyclonal; Promega), rabbit polyclonal anti-cIAP-1 (R and D), mouse monoclonal anti-XIAP (Stressgen), mouse monoclonal anti-GAPDH (Research Diagnostics), and anti-β-galactosidase (mouse monoclonal; Promega).
Techniques: Transfection, Cell Culture, Immunofluorescence, Microscopy, TUNEL Assay, Staining, Plasmid Preparation