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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. 5. — The receptor tyrosine kinase-like orphan receptor ROR2 colocalises and interacts with TMEM67, and is dependent on this interaction for phosphorylation. (A) Four-colour IF imaging showing that endogenous ROR2 (green) colocalizes with TMEM67 (blue) and RPGRIP1L (red) at the ciliary transition zone. Arrowheads indicate regions shown in magnified insets. DAPI is pseudocoloured in grey. Scale bar: 10 μm. (B) Anti-HA co-immunoprecipitations (IPs) demonstrating interaction between full-length exogenous HA-tagged TMEM67 (size 115 kDa) and FLAG-tagged ROR2 (size 105 kDa). Input whole-cell extracts (WCE) for the indicated transfected constructs are on the left. IP of an irrelevant protein (HA-tagged MCPH1) was a negative control. Results are shown for immunoblotting (IB) for anti-FLAG (upper panel) and anti-TMEM67 (lower panel). * indicates a non-specific band in IPs; see supplementary material Fig. S6 for full unprocessed images. (C) Upper panel: IPs demonstrating interaction between FLAG-tagged ROR2 and endogenous TMEM67. Input WCE is shown on the left, and negative control IPs include a no antibody (Ab) control and goat (Gt) and rabbit (Rb) irrelevant (irr.) polyclonal antibodies (PAb). Immunoblotting (IB) for anti-FLAG shows pulldown of FLAG-ROR2 by Gt anti-ROR2 and Rb anti-TMEM67. Lower panel: IPs with irrelevant protein (FLAG-MCPH1, size 93 kDa). (E) Loss of the active phosphorylated ROR2 isoform (labelled P) in mutant Tmem67^−/− cells following Wnt5a treatment, compared with strong induction of the active isoform (upper band, as indicated) in wild-type Tmem67^+/+ cells. Loading control is for β-actin.