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. 2011 Dec 13;287(5):3156–3164. doi: 10.1074/jbc.M111.306019

FIGURE 4.

FIGURE 4.

Direct interaction between ASCIZ and DYNLL1 proteins. A, fluorescence micrographs GFP-ASCIZ-667 localization in human U2OS cells treated with non-targeting control siRNA or Dynll1 siRNA following 4-h treatment with 0.025% MMS. Note that GFP-ASCIZ is retained in the nucleus following DYNLL1 depletion, but fails to form DNA damage-induced nuclear foci. B, schematic diagram of ASCIZ domain topology. ZF, Zn2+ finger; lollipops indicate SQ/TQ (gray) or TQT motifs (green). C, compilation of arbitrarily selected bona fide DYNLL1 binding sites (5–7) (left panel) and eleven ASCIZ TQT motifs numbered in sequential order (compare panel B). D, co-immunoprecipitation (IP) of endogenous ASCIZ and DYNLL1 with ASCIZ but not IgG control antibodies from human U2OS cells. Input represents ∼12.5% of lysate relative to loading of IP lanes. E, co-immunoprecipitation of endogenous DYNLL1 with the ASCIZ antibody from parental (−) or GFP-ASCIZ overexpressing (+) U2OS cells, probed with ASCIZ or DYNLL1 antibodies. Input represents ∼5% of lysate relative to IP lanes. F, co-immunoprecipitation of stably over-expressed GFP-ASCIZ with the FLAG antibody in transiently FLAG-Dynll1 transfected (+) or control (−) cells. Input represents ∼10% of lysate relative to IP lanes. G, fluorescence micrographs of GFP-ASCIZ-667 and FLAG-DYNLL1 in untreated or MMS-treated human U2OS cells; a merged image of the MMS-treated cells is shown underneath. H, yeast two-hybrid assays of the indicated ASCIZ fragments fused to the Gal4-activation domain with or without Dynll1 fused to the Gal4-DNA binding domain vector on selective plates for reporter genes (top) and input control plates (bottom). Western blots of the corresponding cell lysates with ASCIZ and DYNLL1 antibodies are shown below. I, two-hybrid assays similar to panel G, using the ASCIZ TAD fragment as a positive control, the TAD-17AQ mutant as a negative control, and indicated TQT motifs individually restored in the context of TAD-17AQ. 15AQ-TQT#6 + 7 denotes the TAD-17AQ fragment restored with the overlapping TQTQT motifs at positions 6 and 7.