Fig. 4

Nuclear TG2 bound to TOPOIIα after aggregations to DSB sites. A: The workflow of immunoprecipitation and mass spectrometry assays to identify TG2 interacting proteins. B: Venn diagram summarized the results of differentially enriched proteins in irradiated A549 cells, compared with the untreated A549 cells. C: Whole cell lysates were prepared from the A549 cells at 0 and 0.5 h after exposure to γ-irradiation, which were subjected to immunoprecipitation with anti-TG2 antibody and immunoblotted with TOPOIIα primary antibody. D: Whole cell lysates were prepared from the 293T cells transfected with both Flag-tagged TG2 and TOPOIIα constructs after IR (8 Gy) for 30 min, which were subjected to immunoprecipitation with anti-TOPOIIα antibody and immunoblotted with both anti-TG2 and anti-TOPOIIα antibodies. E: In similar, whole cell lysates were also prepared from the 293T cells transfected with FLAG-tagged TG2 and TOPOII α constructs after IR (8 Gy) for 30 min, which were subjected to immunoprecipitation with anti-Flag antibody and immunoblotted with both anti-TOPOIIα and anti-TG2 antibodies. F: Representative images of immunoprecipitation assay for various 293T cells transfected with each TG2 fragments (AB, ABC, AB + C1, BC, CD) and TOPOIIα. G: Representative images of immunoprecipitation assay for various 293T cells transfected with each TG2 mutant fragments (WT, C227S, Y516F, W241A, R580A) and TOPOIIα. Quantification analyses were performed based on the ratio of TOPOIIα and Flag-TG2 mutants density