Fig. 6.

GFP-nSMase2 and V5-nSMase2 co-localize with markers of early and recycling endosomes. (A) MCF-7 cells were plated at low density (0.3 × 10⁶/dish) and 24 h later were transfected with 3′-GFP-tagged nSMase2 (0.5 µg/dish). After 18 to 20 h, cells were treated for 4 h with 50 µg/ml CHX. One hour before the end of treatment, cells were incubated with 10 µg/ml human transferrin-Alexa Fluor 555 (Tfn) or with 1 µg/ml cholera toxin subunit β-Alexa Fluor 594 for the remaining time. Cells were then washed and fixed. Alternatively, cells were washed, fixed, permeabilized, and stained with anti-EEA-1 or anti-transferrin receptor (Tfn-R) antibodies as described in Materials and methods. (B) MCF-7 cells stably overexpressing V5-nSMase2 were transfected with GFP-Rab5 or GFP-Rab11 (0.5 µg/dish). After 24 h transfection, cells were treated with (CHX) or without (none) 50ug/ml CHX for 2 h. Cells were fixed, permeabilized and stained with anti-V5 antibody (green or red) as described in Materials and methods. Co-localization of GFP-nSMase2 with the different markers and of V5-nSMase2 with GFP-Rab5 or Rab11 was analyzed by confocal microscopy. Pictures are representative of at least 4 fields taken from three independent experiments.