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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Nat Neurosci. 2011 Mar 13;14(4):469–477. doi: 10.1038/nn.2781

Fig.2. Flot1 overexpression attenuates Gö6850-mediated inhibition of PKC-triggered endocytosis.

Fig.2

Flot1 overexpression in EM4-YFP-DAT cells attenuated Gö6850-mediated inhibition of PKC-regulated endocytosis. All experiments were performed in this stable cell line unless noted otherwise. a,b. Image based analysis. a. ‘% Cell with Internalization’ was calculated as cells considered positive for internalization (YFP-DAT co-localized to EEA1-positive endosomes) after 1 µM PMA for 30 min. PKC-triggered internalization of DAT was significantly inhibited by Gö6850 in a dose dependent manner (open circles; p < 0.0001). Flot1 overexpression significantly diminished the Gö6850-mediated inhibition (closed circles, p = 0.0078). For each dose, 150 to 200 cells were analyzed. Scale bar = 10 µm. Complete statistics can be found in Supporting Materials. b. Flot1 overexpression in the presence of inhibitor (Flot1+ Gö6850) blunted the effect of inhibitor alone, permitting internalization (white arrows). Scale bar = 10 µm. c. Cell surface biotinylation measuring YFP-DAT surface availability. “DAT surface expression (% vehicle)’ is calculated as described (Fig.1d). Values are normalized to no PMA. Data are plotted as Mean + SEM (n = 5). Ctrl and Gö6850 cells were transfected with mRFP alone as transfection control. Complete blots can be found in Figure. S10.