Fig. 3: The CHAF1A PIP2 domain mediates the CHAF1A-PCNA interaction, but is not necessary for PCNA monoubiquitination.

A Schematics of CHAF1A PIP domains. B Interactions of GFP-CHAF1A (WT, ΔPIP1, and ΔPIP2) with PCNA in HEK293T cells assessed by CoIP using anti-GFP antibody. ΔPIP1: PIP1 domain deletion, ΔPIP2: PIP2 domain deletion. C The binding of CHAF1A mutants to PCNA was detected by in vitro GST pull-down assay. GST-PCNA beads were incubated with lysates of HEK293T expressing GFP-CHAF1A-WT, GFP-CHAF1A-ΔPIP1 or GFP-CHAF1A-ΔPIP2. D The in-situ interaction of GFP-CHAF1A (WT, ΔPIP1, and ΔPIP2) with PCNA in KYSE510 cells was evaluated by PLA assay using GFP and PCNA antibodies. GFP-v was used as a negative control (scale bar, 5 μm). E The number of PLA foci in GFP-positive nuclei was counted, each spot represents the number of PLA foci in an individual nucleus (n = 100). Data are representative of at least three independent experiments. n.s., P > 0.05, ^**P < 0.01; one-way ANOVA. F CHAF1A-knockdown KYSE510 cells were transfected with GFP-CHAF1A-WT or GFP-CHAF1A-ΔPIP2 vectors, and then treated with or without 100 J/m² UV for 1 h. Whole-cell lysates were analyzed using western blotting with the indicated antibodies. G CHAF1A-knockdown KYSE510 cells were transfected with GFP-CHAF1A-WT or GFP-CHAF1A-ΔPIP2 vectors, and then treated with or without 2 mM HU for 4 h. Whole-cell lysates were analyzed using western blotting with the indicated antibodies.