Fig. 3: ARKL1 interacts with RNF111 in a SIM-SUMO dependent manner.
a RNF111 knockdown disrupts the interaction of ARKL1 and BLM. HA IP was performed with lysates of U2OS cells expressing vector, HA-ARKL1 and treated with indicated siRNAs. b Loss of RNF111 abolishes the interaction of ARKL1 and BLM in proximity ligation assay (PLA). Quantifications of WT (n = 170), RNF111 KO (n = 189), ARKL1 KO (n = 115) U2OS cells from two biological replicates are shown. c The SIMs of ARKL1 are required for ARKL1 interaction with both RNF111 and BLM. Lysates from 293 T cells expressing indicated constructs were used. d The co-localization/interaction of RNF111 and ARKL1 is dependent on the SIMs of ARKL1. PLA using antibodies against HA and RNF111 are qunatified for ARKL1 KO U2OS cells complemented with HA-ARKL1WT (n = 28), HA-ARKL1SIM* (n = 20) or vector (n = 17) from two biological replicates. e A schematic of RNF111 deletion mutants generated. f RNF111Δ214–246 deletion mutant exhibits decreased SUMOylation. HA IPs were performed under denature condition using lysates of 293 T cells expressing indicated constructs. g Identification of RNF111 SUMOylation sites at K237 and K238. Sequences flanking the SUMOylation sites from various species are aligned. h RNF111K237/238R mutant exhibits reduced SUMOylation. Flag IP was performed under denature condition using lysates of 293T cells expressing indicated constructs. i The interaction of RNF111 and ARKL1 requires RNF111 K237/238. 293T cells co-expressing GFP-ARKL1 and HA-Flag-tagged RNF111WT or RNF111K237/238R mutant were used. j The co-localization/interaction of RNF111 and ARKL1 requires RNF111 K237/238. PLA using antibodies to HA and ARKL1 are quantified for RNF111 KO U2OS cells complemented with vector (n = 19), HA-RNF111WT (n = 18), and HA-RNF111 K237/238R (n = 28). For b, d, j quantification is shown with mean ± 95% confidence interval (CI), One-way ANOVA with Sidak’s correction was used for statistics, scale bar, 10 μm. Source data are provided as a Source Data file.
