Fig. 2

Nek2 bond β-catenin and regulated ubiquitination and nuclear translocation of β-catenin in HCC cell lines. a. SMMC-7721 cells were transfected with indicated plasmids with or without sorafenib treatment. Flag-tagged protein were precipitated and associated protein were detected with western blotting assay. b. SMMC-7721 cells, transfected with Flag-cmv2 or Flag-Nek2 plasmids, and MHCC-97H cells, transfected with lentivirus containing shRNA for Nek2 were treated with sorafenib for 24 h and analyzed with western blotting assay. c. Flag-Nek2 (green) and β-catenin (red) were detected using immunofluorescence assay. d. SMMC-7721 cells transfected with indicated plasmids for 24 h and sorafenib treatment for another 24 h, then were analyzed with western blotting assay. e, f. SMMC-7721 cells were transfected with Flag, Flag-Nek2, mutation plasmids with sorafenib treatment. Flag-tagged protein were precipitated and associated protein were monitored with western blotting assay. g, h. (Left panels) SMMC-7721 or MHCC-97H stable overexpression or knockdown of Nek2 first treated with sorafenib for 24 h, then treated with CHX (10 μM) and collected cells at indicated timings. (Right panels) Time line charts for β-catenin gray-scale at different timings normalized to β-actin gray-scale. i, j, k. SMMC-7721 and MHCC-97H cells were co-transfected with indicated lentivirus or plasmids with His-tagged ubiquitin in the indicated combinations. Cells were changed with medium containing MG132 (20 μM) 6 h before harvested and analyzed with western blotting assay