Figure 4

DCLK1 overexpression enhances activation of 48-kDa β-catenin via GSK-3β phosphorylation at Ser⁹. (a) Immunoprecipitation of Huh7-RFP-DCLK1 nuclear lysates was carried out with anti-TCF-4 mouse monoclonal (sc-166699) and the blot was probed with anti-ABC 8E7 mAb for active β-catenin. Control immunoprecipitations were carried out with beads alone ((lane 1) and beads plus normal IgG (lane 2) to check specificity of the interaction. (b) β-catenin activity assay using TOP/FOP reporter system (Promega) reflects transcriptional activation of β-catenin/TCF-4-dependent luciferase expression in Huh7-RFP-DCLK1 cells. The cells were co-transfected with plasmids expressing firefly luciferase under the control of wild type (WT) or mutant TCF/LEF binding sites in a promoter and pIS2 plasmid that expresses renilla luciferase (transfection control). Untransfected cells were used as negative control. Luciferase was assayed in 10 μg cell lysates (average of three transfection experiments). (c) Western blot of total lysates prepared from all three cell-types (as indicated) using a mouse monoclonal antibody (mAb) sc373800 (Santa Cruz) that detects phospho-GSK-3β^Ser9 (upper panel). Total GSK-3α and GSK-3β were detected by sc-7291 monoclonal antibody (Santa Cruz). Relative phospho-GSK-3β^Ser9 to actin band intensities are shown at the bottom. These results were confirmed by a similar Western blot analysis. (d) shRNA-mediated downregulation of DCLK1 reduces active 48-kDa β-catenin form. Huh7-RFP-DCLK1 cells were infected with lentiviruses encoding our previously validated anti-DCLK1 shRNA (lane 2) or scrambled shRNA (lane 1)¹⁶. The lysates (30 μg each) were subjected to Western blots for DCLK1, total β-catenin, and active β-catenin.