Fig. 7: POLD3 IS A TARGET OF PARP2. | Nature Communications

Fig. 7: POLD3 IS A TARGET OF PARP2.

From: PARP2 promotes Break Induced Replication-mediated telomere fragility in response to replication stress

Fig. 7

a PARP2-FLAG and E558A cells were transfected with the POLD3-RFP plasmid, and protein expression was analyzed by immunoblotting using a POLD3 antibody. Actin was used as a loading control. b Representative images of RFP:FLAG PLA foci (pink), and antibody controls, detected in PARP2-FLAG and E558A cells. c Quantification of the number of RFP:FLAG foci (pink) per nucleus detected in PARP2-FLAG, and E558A cells after knockdown of BLM with siRNA and overexpression of POLD3. Each dot on the graph corresponds to a specific analyzed nucleus. At least 100 to 300 cells were counted per condition. Red bars represent mean ± SD from n nuclei analyzed from two independent experiments. P-values were obtained using ordinary one-way ANOVA. d Representative images of RFP:FLAG PLA foci (pink) detected in PARP2-FLAG cells after depletion of BLM with siRNA, and inhibition of PARG. e Quantification of the number of RFP:FLAG PLA foci (pink) per nucleus detected in PARP2-FLAG cells after knockdown of BLM with siRNA, and PARG inhibition. Each dot on the graph corresponds to a specific analyzed nucleus. At least 100 to 300 cells were counted per condition. Red bars represent mean ± SD from n nuclei analyzed from two independent experiments. P-values were obtained using ordinary one-way ANOVA. f Heteromodification of POLD3 subunit of human DNA polymerase d by PARP2. Purified Pold was incubated with hPARP2 and activity buffer containing +/− NAD+ and activated DNA. PARylation levels were analyzed by immunoblot with an anti-Poly/mono-ADP ribose antibody. g Representative images of ADPr:POLD3 PLA foci (red) detected in HeLaFAP, PARP1KO, PARP2KO, PARP1/2ko, PARP2-FLAG, and E558A cells after depletion of BLM with siRNA. h Quantification of the number of ADPr:POLD3 PLA foci (red) per nucleus detected in HeLaFAP, PARP1KO, PARP2KO, PARP1/2ko, PARP2-FLAG and E558A cells. Cells were fixed 48 h after the knockdown of BLM with siRNA. Each dot on the graph corresponds to a specific analyzed nucleus. At least 100 to 300 cells were analyzed per condition. Red bars represent mean ± SD. P-values were obtained using ordinary one-way ANOVA. Source data are provided as a Source Data file.

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