Fig. 4: PIN1 promotes SUMO2/3ylation of RNF168 and SUMOylation regulates RNF168 chromatin accumulation. | Nature Communications

Fig. 4: PIN1 promotes SUMO2/3ylation of RNF168 and SUMOylation regulates RNF168 chromatin accumulation.

From: PIN1-SUMO2/3 motif suppresses excessive RNF168 chromatin accumulation and ubiquitin signaling to promote IR resistance

Fig. 4

A HEK293 cells complemented with myc-WT-RNF168 or K210R mutant were transfected with 6xHis-Flag-SUMO2. SUMO2 conjugated proteins were enriched by His-Mag Sepharose Ni beads (Ni2+ pull down) under denaturing conditions and detected by western blotting (Representative of 2 repeats). Source data are provided as a Source Data file. B HEK293 cells containing shRNA control (NTC) or shPIN1 were transfected with myc-RNF168 alone or in combination with 6xHis-Flag-SUMO2. Cells were treated with 1 mM IPTG for 72 hrs for PIN1 depletion. SUMO2 conjugated proteins were enriched by His-Mag Sepharose Ni beads (Ni2+ pull down) under denaturing conditions and detected by western blotting (Representative of 2 repeats). Source data are provided as a Source Data file. C HEK293 cells transfected with myc-RNF168-WT, T208A or T208A/P209A along with 6xHis-Flag-SUMO2. SUMO conjugated proteins were pulled down by His-Mag Sepharose Ni beads (Ni2+ pull down) under denaturing conditions. SUMO-conjugated RNF168 variants were detected by western blotting (Representative of 2 repeats). Source data are provided as a Source Data file. D Western blot of SUMO1 and SUMO2/3 conjugates following siRNA treatments (performed once). Source data are provided as a Source Data file. E U2OS cells stably expressing myc-WT-RNF168 were treated with indicated control and SUMO siRNAs, cells were treated with 2 Gy of IR and fixed after 1 hr post IR and stained for myc and γH2AX. Scale bars 10 µm. F Quantification of myc-RNF168 intensity from (E). Data is mean ± s.e.m, n = 90 cells for siNTC, 110 for siSUMO1 and 146 for siSUMO2/3. Source data are provided as a Source Data file. G Western blot of chromatin fraction and WCL for myc, histone H3, SUMO2/3 and tubulin. Control and SUMO2/3 depleted U2OS cells were treated with 10 Gy of IR and collected for fractionation at indicated time points (performed once). Source data are provided as a Source Data file. H Western blot of chromatin fraction and WCL for myc, histone H3 and tubulin. U2OS cells expressing RNF168-WT, T208A or K210R mutants were treated with 10 Gy of IR and collected at indicated time points for fractionation (performed once). Source data are provided as a Source Data file.

Back to article page