Fig. 4: FBL interacts with PARP1 through the GAR ___domain and is PARylated by PARP1. | Oncogene

Fig. 4: FBL interacts with PARP1 through the GAR ___domain and is PARylated by PARP1.

From: Enhancing low-dose radiotherapy efficacy with PARP inhibitors via FBL-mediated oxidative stress response in colorectal cancer

Fig. 4

A Schematic illustration of bimolecular fluorescence complementation (BiFC) assay (left). FBL was fused to the N-terminal of YFP (YFPn) (FBL-cYFPn). PARP1 was fused to the C-terminal of YFP (YFPc) (PARP1-cYFPc). The images of YFP signal at DNA damage sites induced by 405 nm microirradiation in cells co-transfected FBL-cYFPn with YFPn or PARP-cYFPc, or PARP-cYFPc with YFPn (right) in U2OS cells. B Co-immunoprecipitation of Flag-SBP-FBL with endogenous PARP1 in HEK293T cells. C Co-immunoprecipitation of Flag-SBP-PARP1 with endogenous FBL in HEK293T cells. D Co-immunoprecipitation of Flag-SBP-PARP1 with endogenous FBL in HEK293T cells with or without 1 mM H2O2 treatment for 10 min or 20 min as indicated. E In vitro binding assay for GST-FBL with PARP1. Bacterial purified GST, GST-FBL and GST-FBL ΔGAR proteins were incubated with PARP1 protein overnight and then precipitated with GST sepharose beads. F In vitro binding assay for GST-FBL with PAR polymer. Bacterial purified GST, GST-FBL and GST-FBL ΔGAR proteins were incubated with PAR polymer overnight and then precipitated with GST sepharose beads. G In vitro PARylation assay for GST-FBL by PARP1. H The recruitments of EGFP-FBL-FL, EGFP-FBLΔ21-27 aa (amino acids), EGFP-FBL Δ28-40 aa, EGFP-FBL Δ46-59 aa, EGFP-FBLΔ70-78 aa to the DNA damage sites induced by 405 nm microirradiation in U2OS cells(left). (scale bar =2.5 μm). The quantification of time-lapse recruitments of EGFP-FBL FL and truncations to DNA damage sites induced by 405 nm microirradiation in U2OS (right). (***p < 0.001, error bar =±SEM, n = 10–15 cells). I The schematic illustration for constructing Flag-SBP-Af1521 (Macro ___domain) (top). The immunoblotting analysis of FBL were preformed in Flag-SBP-Af1521 precipitants in HEK293T cells with or without 2 mM H2O2 treatment for 20 min (bottom). The PJ34 (40 mM) was preincubated for 1.5 h before H2O2 treatment. J The schematic illustration of FBL PARylated by PARP1 or recognizing PARylated protein.

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