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. 2016 Feb 4;61(3):434–448. doi: 10.1016/j.molcel.2015.12.017

Figure 1.

Figure 1

IR-Induced Double Phosphorylation of Abraxas C Terminus S404 and S406 Is ATM Dependent

(A) Abraxas-domain boundary and C-terminal sequence containing a serine residue (S404) next to the BRCA1-binding pSPxF motif (high-lighted in blue). The phosphorylation of S404 and S406 is indicated as P.

(B) Double phosphorylation of S404 and S406 residues at the Abraxas C terminus in response to IR in 293T cells and 293T/Abraxas KO cells. The lysates from cells treated with 10 Gy IR followed by incubation at 37°C for 1 hr were used for western blot with anti-pS404pS406 antibody (“∗” non-specific band).

(C) IR-induced double phosphorylation of S404 and S406 is abolished in Abraxas mutants (S404A, S406A, or double mutant, DM) (“∗” non-specific band). The FLAG- and HA-tagged Abraxas WT or mutants were expressed in 293T cells. The lysates from cells treated with 10 Gy IR and incubated at 37°C for 1 hr were used for immunoprecipitation with anti-FLAG beads and western blot with antibodies against pS404pS406, pS406, or HA.

(D) IR-induced double phosphorylation of S404 and S406 occurs immediately after IR treatment. The time points were taken after cells were treated with 4 Gy IR followed by incubation at 37°C.

(E) IR-induced phosphorylation occurs in a dose-dependent manner.

(F) ATM regulates IR-induced phosphorylation. The cells were incubated with ATM kinase inhibitor KU55933 (10 μM) for 2 hr before exposure to 4 Gy IR and subsequent incubation at 37°C for 1 hr.

(G) ATR is not involved in IR-induced double phosphorylation. The ATR inhibitor VE-821 at indicated concentrations was used for treating cells for 2 hr before cells were exposed to 4 Gy IR (see also Figure S1).