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Fig. 2. Expression of EGFP-Myo1b induces the re-distribution of endosomes in the perinuclear region of MNT-1 cells. (A) 20 µg of the membrane-enriched fraction P2, as well as pellets and supernatants that had been collected after high-speed centrifugation of 20 µg P2 treated with 1%Triton X-100 or 1%Triton X-100 with 5 mM ATP for 30 minutes at 4°C, were separated by 7% SDS-PAGE and transferred on nitrocellulose membranes. The membrane was probed with anti-Myo1b antibody (SE7995). Endogenous MYO1B and EGFP-Myo1b are both released after treatment with Triton X-100 supplemented with ATP. (B-J) EGFP-Myo1b-expressing and EGFP-expressing cells (B-G and H-J, respectively) were labelled with (B-D) phalloidin, (E-J) anti-EEA1 antibody and analysed by confocal microscopy. Same confocal section throughout the focal plane of the nucleus for the expression of (B) EGFP-Myo1b and (C) actin, (E) EGFP-Myo1b and (F) EEA1, (H) EGFP and (I) EEA1. (D) Overlay of B and C. (G) Overlay of E and F. (J) Overlay of H and I. The arrows show a membrane area enriched in actin filaments. Notice that overexpression of EGFP-Myo1b affects the cellular distribution of the endocytic compartments. Bars, 2.2 µm (B,E) and 1.9 µm (H).





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