smc5 antibody  (Bio-Techne corporation)

Journal: Nucleic Acids Research

Article Title: RAD18 directs DNA double-strand break repair by homologous recombination to post-replicative chromatin

doi: 10.1093/nar/gkae499

Figure Lengend Snippet: RAD18 promotes recruitment of SMC5 to DNA lesions. ( A ) Relative HR (right) and NHEJ (left) repair in traffic light reporter U2OS cells treated with indicated siRNAs. The repair efficiency was normalized to the total repair efficiency in control siRNA treated cells (mean + SD, n ≥ 3, two-tailed t -test). ( B ) Relative proliferation of parental U2OS and RAD18–KO cells treated with control or 53BP1 siRNA was evaluated using resazurin viability assay 7 days after IR-irradiation with indicated doses (mean ± SD, n ≥ 3, two-way ANOVA). ( C ) Colocalization of SMC5 with γH2AX foci in parental U2OS and RAD18–KO. Cells were incubated with EdU prior IR-irradiation, pre-extracted and fixed at indicated time points. Incorporated EdU was Click-iT-labelled and cells were stained for SMC5 and γH2AX. EdU positive cells are shown (arrows indicate SMC5 foci colocalizing with γH2AX foci, scale bar 10 μm). Right, quantification of cell fraction with >1 SMC5 foci colocalizing with γH2AX foci. EdU positive cells were analyzed (mean + SD, n = 3, two-tailed t -test). ( D ) Quantification of the mean nuclear SMC5 intensity in parental U2OS and RAD18–KO cells. Where indicated, cells were pre-extracted prior fixation (mean ± SD, n = 300, Mann–Whitney test). ( E ) Relative HR and NHEJ repair in traffic light reporter U2OS cells treated with indicated siRNAs. The repair efficiency was normalized to the total repair efficiency in control siRNA treated cells (mean + SD, n ≥ 3, two-tailed t -test). ( F ) Quantification of RAD51 foci in U2OS cells treated with indicated siRNAs for 48 h. Cells were incubated with EdU prior IR-irradiation, pre-extracted and fixed after 6 h. Incorporated EdU was Click-iT-labelled and cells were stained for RAD51. EdU positive cells were analyzed (mean + SD, n = 3, two-tailed t -test). ( G ) Relative proliferation of parental U2OS and RAD18–KO cells treated with control or SMC5 siRNA was evaluated using resazurin viability assay 7 days after IR-irradiation with indicated doses (mean ± SD, n ≥ 3, two-way ANOVA). ( H ) Model describing RAD18 function at DSBs. RAD18 is recruited to the post-replicative chromatin by bimodal recognition of H2AK15Ub and H4K20me0 with its UBZ domain and with ARD domain of its interacting partner SLF1, respectively. RAD18 then inhibits NHEJ by limiting 53BP1 activity and promotes HR by recruiting the SLF2/SMC5/6 complex. RAD18 accumulation at DSBs is inhibited by RAD6-mediated auto-ubiquitination.

Article Snippet: The following antibodies were used in this study: BRCA1 (clone D-9, sc-6954, 1:100 for IF), RAD6 (UBE2A, clone G-9, sc-365507, 1:1000 for WB), Ubiquitin (clone P4D1, sc-8017, 1:500 for WB), PCNA (clone PC10, sc-56, 1:1000 for WB) from Santa Cruz; FLAG (clone M2, F 1804, 1:300 for IF), γH2AX (clone JBW301, 05–636, 1:1000 for WB), 53BP1 (clone BP-13, MAB3802, 1:300 for IF), GFP (clones 7.1 and 13.1, #11814460001, 1:1000 for WB), BrdU (clone BU-33, B8434, 1:100 for IF), anti-conjugated ubiquitin (FK2, 04-263, 1:2000 for IF) from Merck; γH2AX (clone D7T2V, #80312, 1:300 for IF), γH2AX (clone 20E3, #9718, 1:300 for IF), RAD18 (clone D2B9, #9040, 1:400 for IF, 1:1000 for WB), PCNA ubiquityl-Lys164 (clone D5C7P, #13439, 1:100 for IF), H2A (clone D603A, #12349, 1:1000 for WB) from Cell Signaling Technology; 53BP1 (NB100-305, 1:400 for IF), SMC5 (NB100-469, 1:300 for IF, 1:1000 for WB) from Novus Biologicals; RAD51 (ab176458, 1:400 for IF) and H4K20Me0 (ab227804, 1:2000 for IF, 1:1000 for WB) from Abcam.

Techniques: Control, Two Tailed Test, Viability Assay, Irradiation, Incubation, Staining, MANN-WHITNEY, Activity Assay, Ubiquitin Proteomics