GSK1 phosphorylates LIC and reduces its nuclear localization.
(A) The LIC-GFP fusion protein localized in both the nucleus and the cytoplasm (left). LIC-GFP fluorescence intensity was enhanced in the nucleus and weakened in the cytoplasm after treatment with 1 µM 24-eBL (middle). LIC-GFP fluorescence intensity was weakened in the nucleus when co-transformed with GSK1 (right). Numbers in each image show the mean signal intensity (1000×) from at least 10 cells. Data are mean±SE. Bars = 20 µm. (B) Quantification of the fluorescence intensity (105×) and the ratio between the nucleus and the cytoplasm (N/C) in represented areas. N, nuclear signal; C, cytoplasmic signal. (C) Immunoblotting to demonstrate the phosphorylation of LIC by GSK1, which was antagonized by λ-phosphatase 1 (PP1). The level of phosphorylation is shown by autoradiography with an anti-LIC antibody in the top panel and the loaded amount of proteins is indicated by Coomassie Blue staining in the bottom panel. The levels of unphosphorylated LIC relative to the control without GSK1 and PP1 (-P%) were calculated after normalization against the intensity of Coomassie Blue staining and these values are shown beneath the gel images.