Fig. 5: Analytical ultracentrifugation analyses of green fluorescent-tagged Wnt proteins with various sFRPs.

AUC-FDS analysis of the CS of parental (pink; a, e, i), sFRP2- (blue; b, f, g), sFRP3- (green; c, g, k), or sFRP4-(gray; d, h, l) expressing HEK293 cells, co-cultured with GFP-Wnt3a/L (a–d), mClover-Wnt5a/HEK293 (e–h) or Wnt11-mClover/HEK293 (i–l). Peaks indicated by red asterisks correspond those of the Wnt/afamin complex (7.0 S peak, ~150 kD) while a bracket indicates high-molecular-weight (HMW) complexes. Since a small peak at ~4.2 S is detectable even in the CS of normal L cells, this fluorescence appears to be derived from serum components, probably albumin, associated with bilirubin (gray asterisks)³³. In addition, a small peak at 3 S in the CS of Wnt11-mClover/HEK293 cells (i–l) appears due to degradation of Wnt11-mClover (Supplementary Fig. 15). As with GFP-Wnt3a, which forms a 1:1 complex with afamin, as previously reported (a), mClover-Wnt5a (e) also show peaks of the same size (S value), suggesting that mClover-Wnt5a forms a 1:1 complex with afamin. In the presence of sFRP2, these peaks disappear and a new peak (indicated by blue arrowheads) appears at 5.4 S, the size of which corresponds to Wnt/sFRP2 1:1 complex, in the CS of GFP-Wnt3a (b) or mClover-Wnt5a (f), but not Wnt11-mClover (j). Western blotting analysis shows that amounts of sFRP3 and sFRP4 in the CS were equal to or more than that of sFRP2 (Supplementary Fig. 15). Tagged Wnt5a and Wnt11 still retained their activities (Supplementary Fig. 16).