Figure 2

DCLK1 overexpression results in accumulation of 48-kDa hypophosphorylated active β-catenin in hepatoma cells. (a) Huh7-expressing RFP-DCLK1 cells (Huh7-RFP-DCLK1) or Huh7-expressing RFP (Huh7-RFP) were stained with anti-active β-catenin (anti-ABC 8E7 mAb, Millipore) and subjected to confocal microscopy. Blue, nuclear stain with Dapi. Scale bar, 10 μm. (b) Huh7-RFP-DCLK1 and Huh7-RFP cells were similarly immunostained with antibody (AB19022) against total anti-β-catenin that binds C-terminus and detects phosphorylated and unphosphorylated form of β-catenin. (c) Quantitation of nuclear β-catenin in the Huh7-RFP (gray bar) and Huh7-RFP-DCLK1 (hatched bar) using Leica software. The staining intensities for anti-active β-catenin in the nucleus were plotted for both cell-types. p value = 0.0002. (d) A representative Western blot analysis of total lysates (30 μg each) prepared from Huh7-RFP (control, lane 1) and Huh7-RFP-DCLK1 (lane 2) for expression of active and total β-catenin, and its downstream target protein, cyclin D1. Actin, loading control. (e) Detection of cytoplasmic (lanes 1–3) and nuclear (lanes 4–6) levels of active and total β-catenin in parental Huh7 (lanes 1, 4), Huh7-RFP (control, lanes 2, 5), and Huh7-RFP-DCLK1 (lanes 3, 6) cells using Western blot. The blots were probed with antibodies against active and total β-catenin as described above. Mouse mAb (sc-166699) was used for detection of a nuclear protein, TCF-4 that shows enrichment in the nuclear fractions of these cells. Actin and lamin B1, loading controls. The experiment was repeated once to confirmed the results. (f) Active β-catenin (48 kDa) bands in cytoplasmic and nuclear fractions of Huh7-RFP (control) and Huh7-RFP-DCLK1 were quantitated using Gel-Quant. The ratios of active β-catenin to actin were adjusted as 1.0 for the Huh7-RFP lysates and compared with band ratios for Huh7-RFP-DCLK1.