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. 2017 Sep 20;10:296. doi: 10.3389/fnmol.2017.00296

FIGURE 6.

FIGURE 6

Caveolae/Raft-mediated endocytosis is not involved in M6a sorting. M6aGFP-expressing HEK293 cells were subjected to the antibody internalization assay (AIA) in the presence or absence of specific endocytic inhibitors. Methyl-β-cyclodextrin (MCD, 5 mM) and monensin (Mona, 50 μM) were used as inhibitors of clathrin-mediated endocytosis (CME). Filipin (5 μg/ml) was used as inhibitor of caveolae/raft-mediated endocytosis. Transferrin (Tf) 647-conjugate was used to track CME. (A) The panel shows representative confocal images of M6aRFP-HEK293 cells at T1 (1 h after immunouptake) in green, Tf in magenta and surface M6a in gray. (B–D) At least three independent experiments were analyzed for each condition and the percentage of M6a at the cell surface plotted as described in the Section “Material and Methods.” Significant differences were determined using one way ANOVA [F(3,36) = 23.60, p = 0.25] followed by Bonferroni test. (B) T0 vs. T1 ∗∗∗p < 0.001 (n = 11); (C) T0 vs. T1 ∗∗∗p < 0.001 (n = 11); (D) T0 vs. T1 ∗∗∗p < 0.001 (n = 11) and T0 vs. T1 (filipin) ∗∗∗p < 0.001 (n = 9). T0 represents the steady state and T1 1 h after of the immunouptake. Scale bars: 15 μm. Inset: 4 × 4 μm.