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. 2013 Oct 21;42(2):918–925. doi: 10.1093/nar/gkt929

Figure 3.

Figure 3.

S889 phosphorylation regulates the damage-induced phosphorylation of SMARCAL1 in cells without altering its localization. (A) Flag-SMARCAL1 wild-type or S889D proteins purified from HEK293T cells were incubated with lambda phosphatase as indicated, separated by SDS-PAGE and immunoblotted with a total SMARCAL1 antibody. (B) GFP-SMARCAL1 proteins were expressed in HEK293T cells at levels close to endogenous SMARCAL1. Cells were treated with HU for 16 h where indicated and total cell lysates were immunoblotted with SMARCAL1 antibodies. [ΔN = RPA-binding mutant (3)] (C) GFP-SMARCAL1 wild-type, S889A or S889D protein expressing U2OS cells were treated with HU for 5 h, fixed and stained with antibodies to γH2AX. Shown are representative images of cells with co-localized foci. Quantitative measurements found no significant difference in the percentage of cells with GFP-SMARCAL1 S889A, S889D or wild-type foci. (D) Flag-Wild-type, ΔN or S889A SMARCAL1 protein was immunoprecipitated from HEK293T cells. Immunoprecipitated protein complexes were separated by SDS-PAGE and immunoblotted with SMARCAL1 or RPA2 antibodies.