Figure 2

(A) Schematic overview of the targeting approach. Nanobodies were fused to the C terminus of mNphp3(201) for ciliary localization. The protein of interest (POI) is co-expressed with a C-terminal tag or fusion partner that is recognized by the nanobody. Binding of the nanobody to the tag is expected to result in ciliary localization of the POI. (B) Localization of the anti-mCherry nanobody (VHHLaM-2) to primary cilia. mIMCD-3 cells were transfected with mNphp3(201)-VHH_LaM-2-eGFP (green). (C) Localization of the anti-mCherry nanobody and LAPD-mCherry to primary cilia. mIMCD-3 cells were co-transfected with mNphp3(201)-VHH_LaM-2-eGFP (green) and LAPD-mCherry (red). (D) Cytoplasmic localization of LAPD-mCherry. mIMCD-3 cells were transfected with LAPD-mCherry (red). (E) Localization of the anti-mCherry nanobody and bPAC-mCherry to primary cilia. mIMCD-3 cells were co-transfected with mNphp3(201)-VHH_LaM-2-eGFP (green) and bPAC-mCherry (red). All cells shown in B-E were labeled with an Arl13B antibody (cyan, ciliary marker) and DAPI (blue). All scale bars: 10 μm. Boxes indicate the position of the magnified view shown at the bottom right. Arrows in different colors indicate the direction and the length of the shift of the respective fluorescence channel.