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. 2014 Aug 7;95(2):131–142. doi: 10.1016/j.ajhg.2014.06.012

Figure 5.

Figure 5

Coimmunoprecipitation and hTERT-RPE1 Localization Studies of POC1B and FAM161A

(A) Coimmunoprecipitation assay in HEK293T cells. Wild-type 3×HA-POC1B efficiently coprecipitated with 3×FLAG-FAM161A (lane 1), but coprecipitation was reduced for variants 3×HA-POC1B-p.Gln67del and 3×HA-POC1B-p.Arg106Pro (panel 4, lanes 2 and 3). Specificity was confirmed by inclusion of the unrelated p63, which failed to coimmunoprecipitate with wild-type and variant POC1B. As positive controls, coimmunoprecipitation of lebercilin (encoded by LCA5) by FAM161A and of RPGRIP1 by nephrocystin-4 (encoded by NPHP4) was used. Immunoblots of the input are shown in panels 1 and 2, and immunoblots of the FLAG immunoprecipitates are shown in panels 3 and 4. Size markers are depicted in kDa.

(B) Colocalization in hTERT-RPE1 cells. PalmMyr-CFP-FAM161A (green) was targeted to the cell membrane and microtubules and translocated wild-type mRFP-POC1B (red) from the cytosol toward the cell membrane and microtubules.

(C and D) This translocation by PalmMyr-CFP-FAM161A (green) was not observed for mRFP-POC1B-p.Gln67del (C, red) or mRFP-POC1B-p.Arg106Pro (D, red), which both maintained their cytosolic localization. RPGRIP1L (magenta) was used as a transition-zone marker of the cilium. Nuclei were stained with DAPI (blue). Scale bars represent 10 μm. Additional images are shown in Figure S7.