Fig. 1.

The putative PY NLS in GSK-3β and interaction between exogenous GSK-3β and Kap β2. (A) GSK-3β contains the putative-conserved Kap β2 binding motif (¹⁰⁹IVRLRYFFY¹¹⁷) within its binding domain (yellow). Two point mutants were prepared in order to define the binding region. GSK-3β mutants in the Kap β2 putative binding motifs, were also prepared, and the mutated sequences were indicated as Y117A (¹⁰⁹IVRLRYFFY ¹¹⁷ changes to ¹⁰⁹IVRLRYFFA ¹¹⁷) or R111A (¹⁰⁹IVRLRYFFY ¹¹⁷ changes to ¹⁰⁹IVALRYFFY ¹¹⁷). For these mutants control, we used the unrelated GSK-3β K292R mutant. (B) Following immunoprecipitation (IP) using an anti-Kap β2 antibody, an immunoblot (IB) was performed using an antibody against GSK-3β (left). The immunoprecipitated GSK-3β complexes were applied to the immunoblot, using an anti-Kap β2 antibody (right). For the negative control, normal mouse serum was used for immunoprecipitation. (C) Confocal fluorescence micrographs showing the endogenous GSK-3β and Kap β2 in HEK293 cells. Kap β2 was visualized by immunofluorescence in fixed and permeablized cells using polyclonal antibodies to human Kap β2 or GSK-3β and Alexa Fluor 568 conjugated donkey anti-rabbit IgG or Alexa Fluor 488 conjugated mouse anti-rabbit IgG. The yellow pattern resulting from the merging of red and green colors indicates the co-localization of both proteins at a specific region of the nuclear membrane and nuclear. (D) HEK293 cells were transiently transfected with expression vectors, HA-GSK-3β WT, R111A, Y117A. Following immunoprecipitation (IP) using an anti-HA antibody, either Kap β2 (upper lane) or GSK-3β (down lane) was detected with the immunoblot (IB) using an antibody against Kap β2 or GSK-3β. (E) In vitro pull down assay with the fusion protein of GSK-3β (WT, R111A, Y117A, K292R). Whole cell lysates of HEK293 cells was incubated with 1 μg of each glutathione agarose tagged recombinant GSK-3β (WT, R111A, Y117A, K292R). The immunoblot was performed to detect Kap β2 with its antibody (upper lane). The recombinant GSK-3β (WT, R111A, Y117A, K292R) protein amount were monitored with the coomasaie blue staining (bottom lane).