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. 2021 Sep 20;29(3):504–513. doi: 10.1038/s41418-021-00870-4

Fig. 3. Both caspases and proteasome are needed for Otulin degradation.

Fig. 3

A HT1080 cells were treated with the pan-caspase inhibitor Z-VAD-FMK (10 μM) and monitored for Otulin degradation post-SeV infection of 24 h (MOI 10); the treatment with the inhibitor started 1 h before and continued during virus infection. B Otulin degradation was analyzed in HT1080 WT and HT1080 Caspase 3 knockdown (KD) cells post-SeV infection of 24 h. C-caspase 3 was detected as a measure for caspase 3 activation. C Co-immunoprecipitation of Caspase 3 and Otulin in the presence and absence of Z-VAD-FMK during SeV infection for 6 h; the treatment with the inhibitor started 1 h before and continued during virus infection. D HT1080 cells were transfected with poly (I:C) (1 μg) for 24 h to activate RIPA. Z-VAD-FMK treatment started 1 h before transfection and continued throughout the transfection. MG132 (10 μM) treatment was given for 4 h before harvesting cells. Cell lysates were subjected to immunoblot analysis with Otulin antibody to determine full length and cleaved Otulin. Quantification of western blots and statistical analyses are provided in Supplementary Fig. 8 (mean ± SEM, n = 3; ns > 0.05; *P < 0.05; **P < 0.01, ***P < 0.001).