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. Author manuscript; available in PMC: 2019 Jun 14.
Published in final edited form as: Methods Mol Biol. 2016;1454:1–14. doi: 10.1007/978-1-4939-3789-9_1

Figure 2. FRAP to measure protein entry into the primary cilium.

Figure 2.

A-C) FRAP of an NIH 3T3 cell co-expressing the ciliary marker Arl13b-mCherry and the cytosolic kinesin-2 motor KIF17-mCit. A) Whole cell images showing KIF17-mCit (lower left) and Arl13b-mCherry (lower right) expression and the merged channels (top). B) KIF17-mCit fluorescence in the ciliary compartment was photobleached with high laser power and fluorescence recovery was measured every minute for 30 minutes. Dashed white line, ROI for photobleaching. C) Quantification of the fluorescence recovery of KIF17-mCit in the distal tips of cilia (mean ± standard deviation). N = 12 cells. As a cytosolic protein and kinesin motor, the accumulation of new fluorescent KIF17 molecules at the tip of the cilium reflects their entry into the compartment and their movement along the doublet microtubules to the tip. D-F) FRAP of an NIH 3T3 cell co-expressing the ciliary marker Arl13b-mCherry and the peripheral membrane protein RP2-EGFP. D) Whole cell images of RP2-EGFP (lower left) and Arl13b-mCherry (lower right) expression and the merged channels (top). E) RP2-EGFP in the ciliary compartment was photobleached with high laser power and fluorescence recovery was measured every 10 seconds for 3 minutes. Dashed white line, ROI for photobleaching. E) Quantification of the fluorescence recovery of RP2-EGFP in cilia (mean ± standard deviation). N = 11 cells. Scale bar, 5 μm. As a peripheral membrane protein, the recovery of RP2-EGFP fluorescence in the cilium reflects the entry of new RP2-EGFP into the compartment.