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. 2011 Nov 29;287(3):1932–1945. doi: 10.1074/jbc.M111.283457

FIGURE 3.

FIGURE 3.

Ratiometric fluorescence assay to measure RET and TrkB receptor surface levels. A, expression plasmids of RET and TrkB chimeras tagged with Flag epitope were constructed on the pEGFP-N1 backbone as shown. B, receptor cell surface levels were quantified by biotinylation methods in transfected PC12 cells. Immunoreactive bands were quantified by Image J software and cell surface receptor levels were represented as surface/lysate ratios. Relative receptor surface levels were normalized to that of RET-GFP. The results are represented as mean ± S.E. from three independent experiments (**, p < 0.01 versus RET surface levels; Student's t test). C, ratiometric fluorescence assay to quantify RET and TrkB receptor surface levels. PC12 cells expressing indicated receptors were stained with anti-Flag M2 antibody under unpermeabilization condition followed with Alexa Fluor 594-conjugated donkey anti-mouse IgG (red). The Alexa Fluor 594 fluorescence represented surface receptor levels, and the GFP fluorescence represented total receptor levels. Surface receptor levels were represented as the ratios of surface-Alexa Fluor 594/total-GFP fluorescence. Relative receptor surface levels were normalized to that of RET-GFP. The results are represented as mean ± S.E. from three independent experiments (**, p < 0.01 versus RET surface levels; one-way ANOVA).