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. 2013 May 20;288(27):19604–19613. doi: 10.1074/jbc.M112.440388

FIGURE 1.

FIGURE 1.

Identification of a Ku-binding motif in APLF. A, alignment (MultAlin) of human APLF and selected homologues. Invariant residues are identified by red text, and residues with greater than 50% consensus are identified by blue text. B, WCEs from HEK293T cells ectopically transfected with empty vector, HA-APLFWT, HA-APLF1–200, HA-APLF1–180, or HA-APLF1–160 were immunoprecipitated (HA-IP) with anti-HA antibody and immunoblotted with antibodies as indicated. Tubulin was used as a loading control. C, WCEs from HEK293T cells ectopically transfected with empty vector, WT APLF-V5, APLFΔ180–200-V5, or V5-tagged APLF mutants harboring single (R182A, R184A, W189A, M190A, L191A) or combinations of amino acid substitutions (R182A/R184A, W189A/M190A/L191A) were immunoprecipitated with anti-V5 antibody and immunoblotted with antibodies as indicated. D, 50 ng of biotinylated WT- or R184A-APLF, XLF, and WRN peptides was immobilized to streptavidin-Dynabeads, examined for its association with 200 ng of purified Ku70/80 in pulldown assays, and immunoblotted with anti-Ku80 antibody. E, WCEs from HEK293T cells ectopically transfected with empty vector, WT APLF-V5, APLFΔ181–200-V5, or NLS-APLFΔ180–200-V5 were immunoprecipitated with anti-V5 antibody, and the resulting protein complexes were immunoblotted with Ku80, XRCC4, DNA ligase IV, V5, or tubulin antibodies as indicated.