Fig. 4.

Sec15 is required for lysosome exocytosis independently of the exocyst complex. (A) To trigger lysosome exocytosis, HeLa cells silenced with siRNAs targeting the indicated exocyst subunits were treated with 10 μM ionomycin and 4 mM CaCl₂ for 10 min at 37°C. Cell extracts and supernatants were collected and β-hexosaminidase release was quantified as described in the Materials and Methods. Cells transfected with a non-targeting siRNA (siControl) were used as controls. Results were normalized against those of siControl and are represented as mean±s.d. of at least three independent experiments. ANOVA followed by Dunnett's multiple comparisons test was used to compare different data sets with siControl (**P<0.01; all others are non-significant). (B) Co-immunoprecipitation of GFP-Rab11a with Sec15-myc in HeLa cells. Total cell extract (500 μg) was used to immunoprecipitate GFP-Rab11a. Cells expressing GFP were used as a negative control. Input corresponds to 1:10 of total cell extracts used for immunoprecipitation (50 μg). Western blotting was done using mouse anti-Sec15 and goat anti-GFP antibodies. Results are representative of two independent experiments. (C) Representative confocal microscopy images of HeLa cells overexpressing the exocyst subunit GFP-Sec15a (green) and stained with rabbit anti-Rab11a antibody (red). Scale bar: 10 µm. Results are representative of three independent experiments; at least 15 cells were analyzed per experiment.