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. 2015 Jun 1;8(6):527–541. doi: 10.1242/dmm.019083

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — Non-canonical Wnt signalling defects in Tmem67^−/− cells and interaction of Wnt5a with the TMEM67 N-terminal domain. (A) Schematic diagram of conserved domains and structural motifs within the TMEM67 protein, comprising a signal peptide (yellow), a cysteine-rich domain (CRD, orange), regions of β-sheet periodicity (grey), seven predicted transmembrane helices (TM, black) and a coiled-coil domain (CC, blue). Locations are indicated by amino acid residue (aa), with pathogenic missense mutations highlighted in red. The approximate locations of the two epitopes used to raise N-terminal (Nt) and C-terminal (Ct) rabbit polyclonal antibodies (Ab) are indicated. The TMEM67 regions used for exogenous protein expression are indicated by grey boxes. (B) TOPFlash assays to quantify canonical Wnt signalling activity in Tmem67^+/+ and Tmem67^−/− MEFs, following treatment with either control L-cell or Wnt3a-conditioned media, as indicated, and co-transfection with empty vector control, wild-type HA-TMEM67, or HA-TMEM67 containing a series of pathogenic missense mutations. Wild-type HA-TMEM67 rescued de-regulated canonical Wnt signalling in Tmem67^−/− cells, but missense constructs did not. (C) Tmem67^−/− cells had a defective response to Wnt5a, expressed as the ratio of Wnt3a response:combined response to both Wnt3a and Wnt5a. The correct response to Wnt5a was only rescued with wild-type HA-TMEM67. Values shown are means of at least four independent replicates and error bars indicate ±s.e.m. The statistical significance of the pair-wise comparisons with wild-type HA-TMEM67 values (#) are represented as *P<0.05, **P<0.01 and ***P<0.001, Student's two-tailed t-test. (D) Left panel: Coomassie-stained SDS-PAGE analysis of fluorescence-labelled BSA (F-BSA), Wnt3a (F-Wnt3a) and Wnt5a (F-Wnt5a) proteins. Molecular masses of protein size standards (kDa) are indicated. Middle panel: the same gel photographed under UV light to show fluorescent labelling of BSA control, Wnt3a and Wnt5a proteins. Right panel: expression of TMEM67-Nt proteins (predicted molecular mass 48 kDa), containing the indicated missense mutations. (E) Preferential in vitro interaction of F-Wnt5a, but not F-Wnt3a or F-BSA negative control, with increasing amount of wild-type TMEM67-Nt. (F) Interaction of F-Wnt5a with wild-type TMEM67-Nt only, but not TMEM-Nt proteins containing the indicated missense mutations. Values shown are the means of three independent replicates and error bars indicate ±s.e.m. The statistical significance of the pair-wise comparisons with wild-type TMEM67-Nt values (#) are represented as *P<0.05 and **P<0.01, Student's two-tailed t-test.