Journal: The Journal of Cell Biology
Article Title: Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas
Figure Lengend Snippet: FasL induces caspase-dependent cleavage of Grx1 and increases PSSG as well as S-glutathionylation of Fas. (A) Immunoblot analysis of cleaved caspase-8 (p18) and -3 fragments (p17 and p19) in C10 cells treated with FasL + M2 as described in Fig. 1 in the presence or absence of 10 µM ZVAD-FMK. The bottom panel shows total cellular content of Grx1. Note that expression of the pro form of caspase-8 remains unchanged during the course of the experiment. (B) Evaluation of the interaction between Grx1 and caspase-8 or -3 in cells. C10 cells were exposed to FasL + M2 as described in Fig. 1 A , and Grx1 was immunoprecipitated (IP) at the indicated times for the evaluation of association with active caspase-8 or -3 fragments via Western blotting. The bottom panel represents a Grx1 immunoblot. Lanes on the right represent lysates from cells treated with FasL + M2 for 2 h but were subjected to IgG IP as a reagent control. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. The bottom panels represent total content of proteins in whole cell lysates (WCL) that were used as the input for IP. Note that expression of the pro form of caspase-8 remains unchanged during the course of the experiment. Black line indicates that intervening lanes have been spliced out. (C) In vitro assessment of cleavage of Grx1 by caspase-8 or -3. 200 ng recombinant hGrx1 was incubated with 200 U active caspase-8 or -3. At the indicated times, samples were prepared for immunoblot analysis of hGrx1. Fragmented hGrx1 product is ∼8 kD in size. Incubation of heat-inactivated caspase-8 and -3 with hGrx1 for 4 h largely prevented the formation of cleaved fragment (0 h). (D) Increases in overall PSSG are a response to ligation of Fas and are caspase dependent. Cells were incubated as described in A. ZVAD-FMK or vehicle was added to cells 2 h before ligation of Fas as well as 2 h after ligation. Lysates were resolved by nonreducing SDS-PAGE. Antiglutathione antibody was used to detect PSSG on immunoblots. The bottom panel shows total Fas content. (E) Caspase-dependent S-glutathionylation of Fas. C10 cells were incubated with FasL + M2 for 0.5, 1, or 2 h in the presence or absence of ZVAD-FMK. Cell lysates were subjected to nonreducing IP (−DTT) using antiglutathione antibody to IP S-glutathionylated proteins (IP: PSSG) before detection of Fas via Western blotting. As a reagent control to reduce S-glutathionylated proteins before IP, samples were incubated with 50 mM DTT (+DTT). The bottom panel represents Fas content in cell lysates. (F) S-glutathionylation of Fas requires the presence of caspase-8. C10 cells were transfected with control (Ctr) siRNA or caspase-8 (C8)–specific siRNA and 48 h later were incubated with FasL + M2 for 2 or 4 h. The top lane shows assessment of S-glutathionylation of Fas via IP of S-glutathionylated proteins using antiglutathione antibody (IP: PSSG) under nonreducing conditions (−DTT) before detection of Fas via Western blotting. As a reagent control to reduce S-glutathionylated proteins before IP, samples were incubated with 50 mM DTT (+DTT). The bottom panels show total content of Fas, procaspase-8, cleaved caspase-8, cleaved caspase-3, and Grx1 in whole cell lysates. (G) Assessment of caspase-dependent degradation of Grx1 and S-glutathionylation of Fas in NIH 3T3 cells after ligation of Fas. Cells were treated with 500 ng/ml FasL + 1 µg/ml M2 for 1, 2, or 4 h in the presence or absence of ZVAD-FMK. S-glutathionylated proteins were immunoprecipitated as described in E before detection of Fas via Western blotting. The bottom panel represents Fas content, cleaved caspase-3, and Grx1 content in whole cell lysates.
Article Snippet: As a control, a portion of the lysate was treated with 50 mM DTT to reduce glutathionylated proteins, and these samples were purified through columns (Micro-BioSpin; Bio-Rad Laboratories) to remove DTT before subsequent IP.
Techniques: Expressing, Immunoprecipitation, Western Blot, In Vitro, Recombinant, Incubation, Ligation, SDS Page, Transfection