Figure 3.

Casein kinase 2 (CK2) phosphorylates calumenin (Calu)-15 at threonine at position 73 (Thr-73) and facilitates its nuclear localization. (a) The subcellular localization of Calu-15 wild-type (WT), T73A, or T73E. Left panels, representative pictures. Nuclear DNA was stained with DAPI (4',6-diamidino-2-phenylindole; blue). Bar, 10 μm. Right panel, the graph shows the percentage of cells with predominantly nuclear (N>C), both nuclear and cytoplasmic (N=C) or predominantly cytoplasmic (N<C) localization of enhanced green fluorescent protein (EGFP) fusion protein (n=3; >50 cells per experiment). Data are mean±S.E.M.; ***P<0.001; n.s., no significant difference (contingency table test for independence). (b) Lysates of HEK293T cells expressing Calu-15–2 × EGFP WT or T73A mutants (upper panel), and lysates of HEK293T cells expressing Calu-15–2 × EGFP treated with dimethylsulfoxide (DMSO) or 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB; lower panel) were separated by Phos-tag SDS-polyacrylamide gel electrophoresis, then immunoblotted with antibody against EGFP. Arrows indicate the phosphorylated form. (c) HeLa cells transfected with Calu-15–2 × EGFP WT or T73E mutant were treated with DMSO or TBB for 10 h post transfection and then fixed for observation. Left panels, representative pictures. Nuclear DNA was stained with DAPI (blue). Bar, 10 μm. Right panel, the graphs show the percentage of cells with different localization patterns (n=3; >50 cells per experiment). Data are mean±S.E.M.; ***P<0.001; n.s., no significant difference (contingency table test for independence). (d) HeLa cells transfected with Calu-15–2 × EGFP WT, T73A, or T73E were treated with or without leptomycin B (LMB) before fixation and observation. Nuclear DNA was stained with DAPI (blue). Bar, 10 μm. The graphs show the percentage of cells with different localization patterns (n=3; >50 cells per experiment). Data are mean±S.E.M.; n.s., no significant difference (contingency table test for independence)