Supplementary Figure 6: Lysines of 55 and 363 of RIPK3 are responsible to CHIP depletion-induced RIPK3 up-regulation.

(a) 293FT cells were transfected with plasmids expressing the HA-RIPK3 WT or 4KR, K55⋅363R, K89⋅501R mutants in the absence or presence of Myc-CHIP. The lysates were immunoprecipitated using an anti-Myc antibody and then analysed via western blotting. (b) H1299 cells were transfected with HA-RIPK3 WT or K55⋅363R in the absence and presence of FLAG-CHIP, and treated with CHX as indicated. The proteins were detected by WB using anti-HA, -FLAG, -Actin antibodies, quantified by Image J, and are shown in the graph. Half-life of RIPK3 are presented in the graph. (c) H1299 cells were transfected with plasmids expressing GFP/RIPK3 K55⋅363R or K89⋅501R mutants in the absence or presence of FLAG-CHIP. Transfected cells were treated with E64d-Pepstatin A and LysoTracker for lysosome staining. Stained cells were analysed using fluorescence microscopy. A representative image is shown in the upper panel and means from two independent experiments are presented in the lower panel. In each independent experiment, n = 100 cells were counted. (d) Degradation of WT and single point mutant RIPK3 by CHIP was determined by WB in H1299 cells. (e,f) HeLa cells stably transfected with pBabe-RIPK3 WT, K55⋅363R and an empty vector were transfected with the indicated siRNAs for 48 h. Cells were treated with 10 ng/mL hTNFα, 5 μg/mL cycloheximide, and 20 μM z-VAD-fmk (TCZ) for 4 h (e). Cell loss was determined by Cell Titer Glo Luminescent Cell Viability Assay Kit (e), and protein levels were determined by western blot (f). Data are means ± S.D. n = 3 independent experiments (*P < 0.05, **P < 0.01). Raw data from independent experiments are provided in Supplementary Table 1. All scale bars are 10 μm.