Fig. 5: RNF111-mediated ubiquitination of BLM is associated with PML NBs.
a Increased BLM levels in PML NBs in RNF111 and ARKL1 KO cells. BLM intensity in each PML NB in immunofluorescence (IF) was quantified with arbitrary (arb.) units with mean ± 95% CI for WT (n = 1532), ARKL1 KO (n = 1617), RNF111 KO (n = 952) U2OS cells from two biological replicates. One-way ANOVA with Sidak’s correction was used for statistics. b Detection of RNF111 and BLM interaction in PML NBs by PLA in WT or RNF111 KO U2OS cells expressing GFP-PML. RNF111-BLM PLA foci that colocalize with PML NBs are indicated by arrows. c RNF111 SIM domains are required for BLM ubiquitination. The IPs were performed under denature condition using lysates from WT or RNF111 KO U2OS cells expressing indicated constructs and treated with MG132. CHK1 was used as a loading control. d RNF111SIM* mutant fails to reduce the elevated BLM levels in RNF111 KO cells. e RNF111 SIM domains are required for self-ubiquitination. 293T cells expressing indicated constructs were used. f BLM ubiquitination is dependent on its SIMs and SUMOylation at K317/331. GFP IP was performed under denature condition using lysates of 293T cells expressing GFP or GFP-tagged BLM constructs. g ARKL1 SIMs are required for BLM ubiquitination. WT or ARKL1 KO Hela cells expressing indicated constructs were used. In the Input panel, “*” non-specific band; left arrow, endogenous ARKL1; right arrow, Flag-tagged ARKL1. h Expression of WT but not SIM mutant of ARKL1 reduced the elevated BLM levels in ARKL1 KO cells. i RNF111 SUMOylation sites K237/K238 are required for BLM ubiquitination. WT or RNF111 KO U2OS cells expressing indicated constructs were used. Scale bar, 10 μm for a, b. Source data are provided as a Source Data file.
