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Figure 1 | Molecular Cancer

Figure 1

From: Attenuation of WNT signaling by DKK-1 and -2 regulates BMP2-induced osteoblast differentiation and expression of OPG, RANKL and M-CSF

Figure 1

Dkk1 and -2 secreted by MOPC315.4 cells attenuate Wnt3a/β-catenin signaling in L cells. A, Constitutive expression of Dkk1-4 in stable transfectants of mouse plasmacytoma MOPC315.4. Total cell lysate (10 μg) was analyzed by Western blotting using Ab to FLAG, Dkk1-4 or GFP as indicated. β-actin levels (bottom) were determined as loading control. Untreated parental cells and cells transfected with GFP only (control) were included (bottom left). Protein size markers are indicated in kilodalton (kDa) to the left. B, Secretion of Dkk1-4 into the cell culture supernatant. MOPC315.4 transfectants (1 × 104 cells/cm2) were cultured for 3 days and 10 μl CM were analyzed by Western blotting using Ab to His6 and Dkk1-4 as indicated. Note that Dkk2 was secreted as an apparent cleavage product (<17kDa) of the intracellular protein shown in panel A (~40 kDa). C, Dkk1 and -2 inhibit Wnt3a-induced stabilization of β-catenin in L cells. Dkk-CM was added to the cell culture medium at final concentrations of 5, 10, 20, 40 or 80 percent (v/v) as indicated at the bottom. Zero Dkk-CM designates samples supplemented with 20% M-CM. Three hours later, Wnt3a-CM (indicated by plus symbols in the 2nd line from the bottom) or L-CM (no Wnt3a; minus symbols) was added at a final concentration of 10%. Cells were cultured for 3 h and β-catenin (β-cat) and β-actin (β-act) were determined by Western analysis using 10 μg total protein per sample. D, Relative expression levels of β-catenin in L cells exposed to Wnt3a and Dkk1-4. Three immunoblots, including the one presented in panel C, were evaluated by densitometry and the ratio of β-catenin to β-actin was determined. This ratio was defined as one (100%) in cells stimulated with Wnt3a but not exposed to Dkk. Asterisks indicate significant drops in β-catenin in Dkk-treated compared to untreated cells (p < 0.05).

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