Peroxisome targeting in Pex5-depleted and -replenished egg extract. (A) Cleared Xenopus egg extract was incubated with beads containing immobilized, affinity-purified antibodies to Pex5. The depleted extract was subjected to SDS-PAGE, followed by immunoblotting with Pex5 antibodies (lane 2). A control was done with beads lacking Pex5 antibodies (lane 1; mock depletion). In lane 3, 30 nM purified, recombinant Pex5 was added to the depleted extract. (B) Pex5-depleted extract was incubated with 0.6 µM mScarlet-SKL for 1 h at 18°C and imaged with a spinning-disk confocal microscope. (C) As in B, but with mock-depleted extract. (D) As in B, but in the presence of 0.5 µM purified Pex5. (E) As in D, but with additionally added 6 µM cytPex14. (F) As in D, but with 0.5 µM purified Pex5^A510W, a Pex5 mutant defective in SKL binding. These experiments were performed twice. Bars, 10 µm.