Impaired antigen extraction and presentation in Exo70-silenced and GEF-H1 silenced cells. (A and C) Antigen extraction assay. Top: Representative epifluorescence images of control and Exo70-silenced (A) or GEF-H1–silenced (C) cells incubated with BCR ligand⁺ beads coupled to OVA for the indicated times. Cells were fixed and stained for OVA (red) and LAMP-1 (green). Images are shown as Z-projections of a stack. Insets highlight the remaining OVA fluorescence intensity on the bead. Scale bar: 3 µm. Bottom: Quantification of OVA fluorescence intensity remaining on beads. Values were normalized by the initial fluorescence. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; two-way ANOVA with Sidak’s multiple comparison test; Exo70: n ≥ 59 cells from two independent experiments; GEF-H1: n ≥ 73 cells from three independent experiments. (B and D) Antigen presentation assays. Top: Antigen presentation assay with control and Exo70-silenced (B) or GEF-H1–silenced (D) cells. Bottom: Peptide control for the cells used. Mean amounts of IL-2 are shown for a representative of three independent experiments performed in triplicate for Exo70-silenced cells, two independent experiments for GEF-H1–silenced cells. ***, P = 0.0006; ****, P < 0.0001; two-way ANOVA with Sidak’s multiple comparison test. Error bars are mean ± SEM. (E) Representative images of lysosome recruitment to antigen-coated beads in control and GEF-H1–silenced cells. **, P < 0.01; ***, P < 0.001; two-way ANOVA with Sidak’s multiple comparison test; n ≥ 93 cells from three independent experiments.