Figure S2. Characterization of Notum-TM, comparison of Notum and Tiki2, and the effect of Notum on Wnt5a. Related to Figures 2 and 3. This figure provides additional data to support Figure 2 and 3. Appears after Figure 2. (A) Expression of either Notum-TM or Notum inhibited Wnt3a-induced TOPFLASH. Error bars represent SD of triplicated experiments. Notum-TM was more potent than Notum in this assay, while the converse was observed in Figure 2C. These differences were reproducible, and might be explained by differences of the two experimental conditions. Under the condition of this figure Notum-TM was tethered to and concentrated on the cell surface, possibly making it more effective in inactivating Wnt3a near the plasma membrane (where signaling occurs) than Notum, which was diffusible in the CM. On the other hand, under the condition of Figure 2C, Wnt3a CM taken from Notum-expressing cells contained Notum, which likely continued to exert its action after the Wnt3a CM was harvested and applied to responding cells for the duration of the TOPFLASH assay. However Wnt3a CM taken from Notum-TM-expressing cells did not contain Notum (i.e.,Notum-TM), and inactivation of Wnt3a ceased when the CM was harvested, potentially explaining the difference. (B) Notum did not cleave the amino terminal HA tag of HA-Wnt3a, while TIKI2 did. Note that Wnt3a mobility alteration by Notum was much more subtle than that by Tiki2. (C) Coexpression of Wnt5a and Notum resulted in loss of hydrophobicity of the secreted Wnt5a protein, as shown by the Triton X-114 detergent-aqueous phase separation assay, suggesting that Wnt5a is a Notum substrate.
Image published in: Zhang X et al. (2015)
Copyright © 2015. Image reproduced with permission of the Publisher, Elsevier B. V.
Permanent Image Page
Printer Friendly View
XB-IMG-138577