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. 2019 Sep 25;14(9):e0222924. doi: 10.1371/journal.pone.0222924

Fig 4. Identification of NPHP-NUP interactions by the knocksideways assay.

Fig 4

(A) Schematic of the knocksideways assay. NPHP proteins were tagged with mCherry (mCh) and targeted to the mitochondrial surface by fusion to a mitochondrial targeting sequence (mito). The NPHP-mCh-mito bait was co-expressed with an EGFP-tagged nucleoporin (GFP-Nup). An interaction between the NPHP and nucleoporin proteins recruits the GFP-Nup to the mCh-labeled mitochondrial surface. (B-D) NPHP2-mCh-mito recruits the inner ring nucleoporins (B) GFP-Nup35 and (C) Nup155-EGFP3 and the outer ring nucleoporin (D) GFP-Nup133 to the mitochondrial surface. (E-G) NPHP5-mCh-mito recruits the inner ring nucleoporins (E) GFP-Nup35 and (F) Nup155-EGFP3 and the outer ring nucleoporin (G) GFP-Nup133 to the mitochondrial surface. Scale bars, 10 μm. Yellow text indicates the number of cells positive (pos.) for a knocksideways interaction. (H) Summary of the results. +, recruitment of GFP-Nup observed in >50% of cells; +/-, recruitment of GFP-Nup observed in 10–50% of cells; -, recruitment of GFP-Nup observed in <10% of cells.