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. 2019 Apr 10;218(6):2021–2034. doi: 10.1083/jcb.201901152

Figure 3.

Figure 3.

Protein targeting to peroxisomes depends on Pex5 and Pex14. (A) Cleared Xenopus egg extract was incubated for 1 h at 18°C with 0.5 µM GFP-SKL in the presence of 6 µM of a cytosolic fragment of the peroxisome docking protein Pex14 (cytPex14). The sample was imaged with a spinning-disk confocal microscope. (B) As in A, but without adding cytPex14. (C) Egg extract was incubated with beads containing immobilized cytPex14 and subjected to SDS-PAGE, followed by immunoblotting with Pex5 antibodies (lane 2). A control was done with beads lacking cytPex14 (lane 1; mock depletion). Purified, recombinant Pex5 was analyzed either without added extract (lane 3) or after addition of different amounts to depleted extract (lanes 4–6). MW, molecular weight. (D) Pex5-depleted extract was incubated for 1 h at 18°C with 0.5 µM GFP-SKL and imaged with a spinning-disk confocal microscope. (E) As in D, but with mock-depleted extract. (F) As in D, but in the presence of 1 µM purified Pex5. (G) As in F, but with 1 µM purified Pex5A510W, a Pex5 mutant defective in SKL binding. All experiments were performed at least three times. Bars, 10 µm.