Fig 1: Mad2 phosphorylation impaired DSB repair. (A,B) Homology-directed repair assay in control shRNA or ATM shRNA cells subjected to vector, WT, S195A, or S195D transfection. (C,D) Nonhomologous end-joining assay in control shRNA or ATM shRNA cells subjected to vector, WT, S195A, or S195D transfection. (E) Colony formation assay of control shRNA or ATM shRNA cells transfected with empty vector only, WT, S195A, or S195D with or without interventional radiology treatment. Mean ± SD, Student's t-test, n = 3, *p < 0.05. (F) CCK-8 assay analysis of cell proliferation abilities in control shRNA or ATM shRNA cells subjected to vector, WT, S195A, or S195D transfection.
Fig 2: E2 interacts with regulators of mitosis completion including the mitotic checkpoint complex, the spindle itself and the kinetochores. (A) Table listing the interactants of E2 associated with regulation of mitosis completion. See also Table S1. (B) Immunofluorescence showing co-localization of GFP-E2 with SAC proteins (BUBR1 and MAD2), kinetochores (CENP-E) and mitotic spindle (β-tubulin). (C) Immunoprecipitation confirming interactions of GFP-E2 with Cdc20, BUBR1 and MAD2 (I = Input, B = Beads).
Fig 3: Elimination of ATM increased phosphorylation of Mad2. (A) Immunoblotting analysis of control or ATM shRNA cells in absence or presence of nocodazole. (B) Immunoblotting analysis in simian virus 40-transformed fibroblast cell lines GM9607 and GM0637. (C) Immunoblotting analysis of cells treated with mock or nocodazole in presence or absence of λPPase. (D) Immunoblotting analysis of HeLa cells transfected with vector, WT, S214A, or S214D mutant form of Mad1 of in presence or absence of nocodazole.
Fig 4: Mad2 is mainly phosphorylated at Ser195 in ATM-loss cells. (A) Immunoprecipitation assay of detecting abundance of Mad2 phosphorylation in control or ATM shRNA cells. (B) Some phosphorylated sites in Mad2. (C) Structures of Mad2 S120, S170, S178, S185, and S195 are shown as ball-and-stick. (D) Immunoprecipitation assay of HeLa cells transfected with GFP-tagged WT, S170A, S178A, S185A, or S195A mutants of Mad2. Exogenous proteins were immunoprecipitated with anti-GFP antibody followed by immunoblotting using indicated antibodies. Statistic analyses were done by t-test, and p values are presented.
Fig 5: Phosphorylation of Mad2 Serine 195 regulated Spindle Checkpoint. (A) FACS analysis of HeLa cells transfected with vector, WT, S195A, or S195D mutant form of Mad2 and treated with nocodazole followed by flow cytometric anti-phospho-H3 staining. (B) Mean mitotic percentages (at least triplicate samples) and error bars represent variations around averages. (C) Co-immunoprecipitation assay of Mad2 complex in 293FT cells transfected with GFP-tagged S195A or S195D mutants of Mad2.
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