Fig. 2.
V5-nSMase2 localizes in the Golgi and at the plasma membrane after inhibition of protein synthesis with cycloheximide. MCF-7 cells stably overexpressing V5-nSMase2 were seeded at 0.1 × 106/dish in 35-mm plates and 24 h later were treated with or without 50 µg/ml cycloheximide (CHX) for the indicated time (A) or for 2 h (B). (A) Total protein was extracted and immunoblotted with anti-V5 antibody as described in Materials and methods. The intensity of bands was evaluated with ImageJ software. Results are representative of three independent experiments. (B) Cells were fixed, permeabilized, and stained with anti-V5 (green) and anti-Giantin (red) antibodies as described in Materials and methods for analysis by confocal microscopy. Pictures are representative of at least 4 fields taken from three independent experiments.