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. 2011 Feb 28;286(16):14237–14245. doi: 10.1074/jbc.M110.165464

FIGURE 5.

FIGURE 5.

NPHP4 controls the subcellular localization of NPHP1 in human ciliated retinal pigment epithelial cells. A, characterization of a novel murine monoclonal antibody detecting the N terminus of NPHP1. FLAG-tagged NPHP1 and NPHP1237–670 or NPHP4304–950 were expressed in HEK293T cells. Immunoblotting (IB) with an anti-FLAG antibody revealed expression of the indicated proteins. The novel NPHP1 mAb detected full-length NPHP1 but neither NPHP4304–950 nor NPHP1237–670. B, characterization of NPHP4 siRNA. Plasmids for F.NPHP4 and F.Eps1–225 (epidermal growth factor receptor pathway substrate 15-like 1) and the indicated siRNAs were co-transfected in HEK293T cells. NPHP4 siRNAs 2, 3, and 4 specifically reduced NPHP4 expression. C, NPHP4 knockdown leads to an accumulation of NPHP1 in the trans-Golgi network. hTERT-RPE1 were transfected with either control siRNA or siRNA directed against NPHP4. After methanol fixation immunofluorescence staining was performed with the indicated antibodies in the absence of Triton X-100. Transfection of NPHP4 siRNA led to an increased NPHP1 signal co-localizing with a staining of the trans-Golgi marker Golgin-97. All RNAi experiments were performed three times.