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. Author manuscript; available in PMC: 2021 Mar 9.
Published in final edited form as: Cell Rep. 2021 Jan 26;34(4):108680. doi: 10.1016/j.celrep.2020.108680

Figure 1. ASF1A phosphorylation at Ser-166 is required for its interaction with MDC1 on DSBs.

Figure 1.

(A) Poor conservation of C-terminal ASF1A and ASF1B in humans, and C-terminal deletion mutants of ASF1A with HA tag used in (C).

(B) MDC1 interaction with ASF1A, not ASF1B, in presence of DSBs. HA-tagged ASF1A or ASF1B was immunoprecipitated with anti-HA antibody conjugated beads in HEK293T cells after 20 µg/mL bleomycin treatment for 1 h. Shown are immunoblots of immunoprecipitates and lysates. EV, empty vector; 1A, HA-ASF1A; 1B, HA-ASF1B.

(C) 160–170 amino acids of C-terminal ASF1A required for its interaction with MDC1. Immunoprecipitation (IP) was performed as described in (B).pATM, a marker for DSBs.

(D) Phospho-mimetic mutation of Ser-166 in ASF1A facilitates its interaction to MDC1. The wild-type (WT), Ser166Ala (S166A) or Ser166Glu (S166E) mutant of HA-ASF1A was applied to the IP.

(E) In vitro binding of the phospho-mimetic ASF1A to recombinant FHA domain of MDC1. Recombinant GST or GST-tagged FHA domain of MDC1 (aa 1–158) was incubated with recombinant WT or S166 mutants of his-tagged ASF1A and then immunoprecipitated by glutathione beads followed by immunoblotting against the indicated antibodies.