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Fig. 9. EGFP-Myo1b and Pmel17 associated to magnetic-endosome co-immunoprecipitate. (A) Post-nuclear supernatant (PNS), non-magnetic (NMF) and magnetic fractions (MF) isolated from wild-type or EGFP-Myo1b cells as described in Materials and Methods, were analysed by SDS-PAGE, and western blotting with anti-Rab5 and anti-Rab7antibodies in the case of wild-type cells, anti-Myo1b (SE7995) and anti-Pmel17 (Pep13h) antibodies for the magnetic fraction of both cell types, and anti-GFP antibodies for the magnetic fraction isolated from EGFP-Myo1b cells. Note that MYO1B as well as P1, P2 and Mß polypeptides of Pmel17 are present in the magnetic fraction enriched for Rab5, and that EGFP-Myo1b is detected on the magnetic fraction isolated from EGFP-Myo1b cells. (B) Lysate of the magnetic fraction isolated from EGFP-Myo1b cells was immunoprecipitated with anti-GFP antibody and analysed by SDS-PAGE, and western blotting with anti-Pmel17 (Pep13h) antibody. The molecular mass of the immunoprecipitated proteins was compared to the molecular mass of the Pmel17 precursor forms P1 and P2, which had been detected with the same antibody in the post nuclear supernatant (PNS). A polypeptide of the size comparable with P2 co-immunoprecipitates with EGFP-Myo1b.





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