Fig. 1. Expression of 6His-tagged Dkk1 protein. (A) Histogram reporting for each cell line the 6His-tagged Dkk1 expression levels in CM estimated by semi-quantitative dot blot. (B) Heparin addition to the culture medium increased recovery of 6His-tagged Dkk1. CM hundred microliters from three different 6His-tagged Dkk1 expressing clones (A–C) cultured with or without heparin at 0.1 g/L was analyzed by dot blot using anti-tetra His antibody (Qiagen). Known amounts of purified GST-6His-RANK protein were loaded for quantification purposes. (C) Analysis of 6His-tagged Dkk1 expression using 10–20% SDS–PAGE stained with Coomassie blue using 12.5-fold concentrated CM. Lanes 1–3: 0.2, 1 and 2 μg BSA (MW: 66 kDa). Lanes 4–6: 5, 10 and 20 μl of 12.5-fold concentrated CM from FreeStyle-293F parental cells. Lanes 8–10: 5, 10 and 20 μl of 12.5-fold concentrated CM from 3F8 clone cells. Lane 7: SeeBlue molecular weight markers.

Fig. 2. Purification of 6His-tagged Dkk1 protein. (A) Nickel chromatography: samples were separated on 10–20% SDS–PAGE and revealed by Coomassie blue staining. Loading volumes: 20 μl for (A–E), 4 μl for the elution fractions (A8–B10). Lane A: Pool of CM; lane B: pool of CM, clarified by centrifugation at 16,000g for 3 h; lane C: as in (B) + buffer containing 20 mM imidazole; lane D: pellet obtained after centrifugation (10 times concentrated); lane E: flowthrough of the nickel column; lanes A8–B10: fractions obtained by imidazole gradient elution. (B) Concentrated 6His-tagged Dkk1 was analyzed using 10–20% SDS–PAGE followed by Imperial blue staining. Lanes 1 and 2: 20 and 10 μl of purified protein before concentration; lanes 3 and 4: 5 and 2.5 μl of purified protein after concentration.

Fig. 3. Activity of purified 6His-tagged Dkk1 protein. (A) Fibroblast-L-cells were transiently transfected with TOPflash reporter as described under Materials and methods and treated in the presence or absence of Wnt3a-CM with increasing amounts of 6His-tagged Dkk1 protein. Luciferase activity was measured as described in Materials and methods. (B) C3H10T1/2 cells were grown in the presence or absence of Wnt3a-CM and treated with increasing amounts of 6His-tagged Dkk1 protein. ALP activity was measured as described in Materials and methods. (C) CM containing either EC-LRP5 or EC-LRP6 protein was incubated with 6His-tagged Dkk1 followed by immunoprecipitation with anti-V5 antibody. Co-precipitation of the EC-LRPs with 6His-tagged Dkk1 (also V5-tagged) protein was revealed by Western blotting using anti-V5 and anti-Myc antibodies.

Fig. 4. Oligomerization studies of 6His-tagged Dkk1 protein. (A) Chromatogram of Superdex 200 HR10–30 (24 ml) gel filtration (UV_280nm) performed on 6His-tagged Dkk1 protein eluted from nickel chelate column. Injection: 0.5 ml. Flow rate: 0.4 ml/min. (B) Elutions fractions from P2 and P3 gel filtration peaks were subjected to 10–20% SDS–PAGE followed by Coomassie blue staining. Ten microliters of each fraction was loaded. (C) Fibroblast L-cells were transiently transfected with TOPflash reporter and grown in the absence (Ctrl) or presence of (others) Wnt3a-CM. Increasing amounts (25–250 ng/ml) of 6His-tagged Dkk1 protein from fractions “start, P1, P2 and P3” were added to the culture medium. Luciferase activity was measured as described in Materials and methods.

Fig. 5. Glycosylation studies of 6His-tagged Dkk1 protein. (A) Inhibition of 6His-tagged Dkk1 glycosylation in vivo by tunicamycin treatment. Samples were separated on 10–20% SDS–PAGE, transferred onto nitrocellulose filters followed by detection with the anti-tetra His antibody. Loading: 16 μl of CM from 3F8 clone grown for 24 h in the presence of indicated amounts of tunicamicyn. (B) Deglycosylation study of 6His-tagged Dkk1. 6His-tagged Dkk1 protein was treated with PNGase F and neuraminidase at 37 °C followed by centrifugation and 10–20% SDS–PAGE/Coomassie blue analysis. Lane 1: no enzyme (6 h); lanes 2 and 3: PNGase F (3 and 6 h, respectively); lane 4: PNGase F (3 h) followed by neuraminidase (3 h); lanes 5 and 6: neuraminidase (6 and 3 h, respectively); lane 7: neuraminidase (3 h) then addition of PNGase F (3 h); lanes 8 and 9: Neuraminidase and PNGase F (3 and 6 h, respectively). (C) Deglycosylated proteins still inhibit Wnt signaling. Fibroblast-L-cells were transiently transfected with TOPflash reporter and grown in the presence of Wnt3a-CM and increasing amounts (25–1000 ng/ml) of deglycosylated 6His-tagged Dkk1 protein. Luciferase activity was measured as described in Materials and methods and percent of Wnt transduction pathway inhibition is reported.

6His-tagged Dkk1 protein purification table