Casp8p41 facilitates Bak oligomerization and Bak-mediated MOMP. (A) Dextran-FITC–loaded liposomes composed of MOM lipids were incubated with 50 nM His6-BakΔTM in the absence or presence of 200 nM GST, GST-Casp8p41, or GST-Casp8p41 V150E/L157K at 37°C for 30 min. Bim BH3 peptide served as a positive control. After bismaleimidohexane cross-linking, samples were subjected to SDS-PAGE and blotted for Bak. (B) After His6-BakΔTM was incubated with GST or GST-Casp8p41 in buffer containing 1% (wt/vol) CHAPS, fractions from size exclusion chromatography were blotted for Bak and GST. Representative of n = 3. (C) FITC-dextran release over time from FITC-dextran–loaded liposomes treated as in A. Representative of n = 3. (D) Summarized results from three independent experiments at 300 s of FITC-dextran release from liposomes. (E) FITC-dextran release from liposomes treated with 50 nM His6-BakΔTM in the presence of 200 nM GST-Casp8p41, GST-Casp8p41 V150E/L157K (EK), or GST. 100% release in D and E was determined by treating liposomes with Triton X-100. Error bars indicate ±1 SD from three independent experiments (*, P < 0.05; ***, P < 0.001). (F–J) After mitochondria (F) from wt (G), Bax−/−Bak−/− double knockout (H), Bax−/− (I), or Bak−/− (J) MEFs were incubated with 200 (1), 500 (2), or 1,000 nM (3) of purified protein for 1 h, sedimented, and washed, the supernatants and pellets were blotted for cytochrome c (Cyto C) and, as a control, the mitochondrial matrix protein Hsp60. Bim BH3 (200 nM), which induces CytoC release in the presence of either Bak or Bax, served as a positive control. Representative of n = 3.
Casp8p41 facilitates Bak oligomerization and Bak-mediated MOMP. (A) Dextran-FITC–loaded liposomes composed of MOM lipids were incubated with 50 nM His6-BakΔTM in the absence or presence of 200 nM GST, GST-Casp8p41, or GST-Casp8p41 V150E/L157K at 37°C for 30 min. Bim BH3 peptide served as a positive control. After bismaleimidohexane cross-linking, samples were subjected to SDS-PAGE and blotted for Bak. (B) After His6-BakΔTM was incubated with GST or GST-Casp8p41 in buffer containing 1% (wt/vol) CHAPS, fractions from size exclusion chromatography were blotted for Bak and GST. Representative of n = 3. (C) FITC-dextran release over time from FITC-dextran–loaded liposomes treated as in A. Representative of n = 3. (D) Summarized results from three independent experiments at 300 s of FITC-dextran release from liposomes. (E) FITC-dextran release from liposomes treated with 50 nM His6-BakΔTM in the presence of 200 nM GST-Casp8p41, GST-Casp8p41 V150E/L157K (EK), or GST. 100% release in D and E was determined by treating liposomes with Triton X-100. Error bars indicate ±1 SD from three independent experiments (*, P < 0.05; ***, P < 0.001). (F–J) After mitochondria (F) from wt (G), Bax−/−Bak−/− double knockout (H), Bax−/− (I), or Bak−/− (J) MEFs were incubated with 200 (1), 500 (2), or 1,000 nM (3) of purified protein for 1 h, sedimented, and washed, the supernatants and pellets were blotted for cytochrome c (Cyto C) and, as a control, the mitochondrial matrix protein Hsp60. Bim BH3 (200 nM), which induces CytoC release in the presence of either Bak or Bax, served as a positive control. Representative of n = 3.
