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. 2017 Jan 6;24(4):615–625. doi: 10.1038/cdd.2016.153

Figure 5.

Figure 5

Sendai virus proteins restrict cIAP1-mediated RIP1 ubiquitination. (a) Knockdown of CYLD protein by siRNA in L929 cells rescues SeV induced necroptosis. Cells transfected with control siRNA or CYLD siRNA were treated with the indicated agent and/or SeV. Right panel: western blot analysis of the control siRNA or CYLD siRNA treated cells using CYLD- or β-actin specific antibodies. (b) Knockdown of CYLD using shRNA in 293T cells overexpressing indicated constructs. RIP1 was immunoprecipitated (RIP1 IP) from 1% NP40 lysates and its ubiquitination was monitored by western blot with ubiquitin-specific antibodies. Lower panel: western blot analysis of lysates with the indicated antibodies. (c) Left panel: treatment of L929 cells with 100 nM SMAC Mimetic (SM) and infection with SeV (10 HA U/ml) causes death of cells. Right panel: SMAC mimetic treatment sensitizes low dose zVAD (2.5 μM) and SeV infection to cell death. (d) RIP1 was immunoprecipitated from 1% NP40 lysates generated from 293T cells transfected with indicated constructs. cIAP1-mediated RIP1 ubiquitination was monitored by western blot. Top panel: RIP1 immunoprecipitates (RIP1 IP) were analysed by western blot analysis using ubiquitin- or RIP1-specific antibodies. Lower panel: lysates from the transfected cells were analysed using the indicated antibodies. Asterisk indicates non-specific band. (e) Top panel: loss of cIAP1 protein expression upon infection with SeV after 10 h post-infection (hpi). Lower panel: lysates from L929 cells treated with indicated agents were resolved by SDS-PAGE and western blot analysis confirmed loss of cIAP1 protein in wild type (WT) SeV-infected samples but not d2Y mutant-infected samples. (f) L929 cells were transfected with pCI or Y1/Y2 constructs and subsequently treated with zVAD (2.5 μM) and SM (100 nM). Cell death was determined by 7AAD+ staining by flow cytometry. For, 5A, 5C, 5F, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. All experiments repeated at least three times. ***P<0.001, **P<0.01, *P<0.05