Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection

Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K.; Liebert, Uwe G.

Nuclear fractionation was performed by incubation of confluent cells grown in a 60 mm dish (initially 1 ¢ 10 6 cells were plated) in 500 µL buffer A (10 mM HEPES, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.05% NP-40, pH 7.9) supplemented with 1 mM PMSF and protease inhibitor mix for 15 min on ice under shaking conditions. Lysed cells were centrifuged at 3000 rpm and 4 ¥ C for 10 min. The pellet was resuspended in 94 µL buffer B (5 mM HEPES, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, 26% (v/v) glycerol pH 7.9) supplemented with 1 mM PMSF and protease inhibitor mix. Each sample was charged with 6 µL 5 M NaCl and incubated under shaking conditions for 2 h at 4 ¥ C. Finally, samples were centrifuged at 13,000 rpm for 20 min at 4 ¥ C and the supernatant was collected as nuclear fraction. Mitochondria-enriched fractions were obtained by differential centrifugation as published [11] . Mitochondrial (30 to 120 µg/lane) and nuclear (20 µg/lane) fractions were subjected to SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane for detection with primary (anti-VDAC antibody was diluted 1:2000, all other antibodies 1:200) and HRP-conjugated secondary antibodies as described [11] . Equivalent protein loading in the mitochondrial and nuclear fraction was monitored with VDAC and histone 2B (H2B), respectively.