Supplementary Figure 7: The K571⋅604⋅627R (3KR) RIPK1 are resistant to degradation and lysosomal localisation by CHIP.

(a) H1299 cells were transfected with HA-RIPK1 mutants with or without FLAG-CHIP as indicated. Degradation of each mutant by CHIP were determined by WB. (b) The amino acid sequence alignment of human and mouse RIPK1. (c) 293FT cells were transfected with plasmids expressing the HA-RIPK1 WT or 3KR mutant in the absence or presence of Myc-CHIP. The lysates were immunoprecipitated using an anti-Myc antibody and then analysed via western blotting. (d) 293FT cells transfected with the plasmid expression HA-RIPK1 (WT and 3KR) and His-Ub with or without FLAG-CHIP in the presence and absence of E64d-Pepstatin A. A pull-down assay was performed using Ni²⁺-NTA beads. The level of RIPK1 ubiquitylation was determined by WB using an anti-RIPK1 antibody. (e) H1299 cells were transfected with a combination of GFP/RIPK1 WT or 3KR and FLAG-CHIP and were treated with E64d/Pepstatin A and LysoTracker. The transfected cells were stained using an anti-FLAG antibody and then analysed via fluorescence microscopy. A representative image is shown in the left panel and means from two independent experiments are presented in the right panel. In each independent experiment, n = 100 cells were counted. All scale bars are 10 μm.