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. 2014 Oct 15;34(42):13976–13987. doi: 10.1523/JNEUROSCI.1938-14.2014

Figure 7.

Figure 7.

Numb interacts with Cng channel subunits and promotes trafficking of Cnga1 to the recycling endosome. A, Confocal z-stack projection of a cDKO retinal section at P60 immunostained for Cnga1 (red) and Hoechst (blue). The dotted line indicates the XZ slice view shown in A′. Cnga1 accumulates on the outer edge of the IS (arrow). B–E, Electron micrographs of control (B, C) and cDKO (D, E) photoreceptors immunostained for Cnga1 at P60. DAB precipitates were silver intensified and appear as black dots on the image. The boxed areas in B and D indicate the magnified views shown in C and E. Cnga1 labeling is indicated by arrows. F, Coimmunoprecipitation of Cnga1 and Numb in HEK293 cells. G, Coimmunoprecipitation of Cnga1 and Numb in vivo at P60. H, Coimmunoprecipitation of Numb and Cngb1 in vivo at P60. I, Pull down of Cnga1 with Ubiquitin-HA (Ubi::HA) in HEK293 cells, in presence of exogenous Numb (Numb::Myc) or not. IB, Immunoblotting. J–O, Cos-7 cells cotransfected with Rab11::DsRed and mouse Cnga1, in the presence of exogenous Numb (Numb::myc) or a control empty construct (myc). Cnga1 was detected by immunostaining. The amount of Cnga1 in the recycling endosome (arrowheads) is increased upon Numb overexpression. P, Fluorescence intensity measurements of the percentage (%) of Cnga1 signal localized in the Rab11 compartment over the total Cnga1 signal in control transfected (myc) and Numb overexpressing (Numb::myc) Cos-7 cells (mean ± SEM; myc: n = 30 cells; Numb::myc: n = 28 cells; ***p = 0.002, Student's t test). Q, Fluorescence intensity measurements of the percentage (%) of Ezrin::GFP signal localized in the Rab11 compartment in control transfected (myc) and Numb overexpressing (Numb::myc) Cos-7 cells (mean ± SEM; myc: n = 17 cells; Numb::myc: n = 20 cells; ***p = 0.52, Student's t test). R, A model of Numb/NbL function in photoreceptor cilia. In wild-type photoreceptors (left), Numb interacts with the Cng channels post-Golgi and promotes their trafficking to the recycling endosome, where they are sorted for specific delivery to the OS plasma membrane. In the absence of Numb (right), the Cng channel is not trafficked to the recycling endosome and cannot be sorted, such that it ends up localizing to both the IS and OS plasma membrane.