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Quantitation of GLUT4 plasma membrane insertion.

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posted on 28.01.2016, 03:11 by Verena Stadlbauer, Renate Haselgrübler, Peter Lanzerstorfer, Birgit Plochberger, Daniela Borgmann, Jaroslaw Jacak, Stephan M. Winkler, Klaus Schröder, Otmar Höglinger, Julian Weghuber

(A) Representative TIRF microscopy images and fluorescence intensity profiles for the indicated line scans (marked in yellow in the merged images) of GLUT4-GFP and Alexa647-anti-myc staining under basal and compound-treated conditions. CHO-K1 hIR/GLUT4-myc-GFP cells were grown over night in 96-well plates (35,000 cells/well), starved for 3 hours in HBSS buffer, stimulated for 10 minutes, fixed with 4% para-formaldehyde, and stained using an Alexa647 labeled anti-myc antibody. Fluorescence in the evanescent field was recorded for GFP and Alexa647 at 488 and 640 nm, respectively. Scale bar = 20 μm. (B) Quantitation of fluorescence signals in stimulated cells with respect to untreated cells (n > 120 cells). Fluorescence was normalized to the values prior stimulation. Error bars are based on the standard error of the mean. ***P < 0.001 and ****P < 0.0001, significant increase with respect to starved cells. (C) Binding probability determined by fluorescence-guided AFM measurements under starved and compound treated conditions. Cells were grown on standard 30 mm glass slides (350,000 cells/slide) over night, starved for 3 hours in HBSS buffer, stimulated with the indicated substances for 10 minutes, and fixed with 4% para-formaldehyde. Error bars are based on the standard error of the mean. ****P < 0.0001, significant increase with respect to starved cells. (D) Linear correlation regression analysis between myc-staining and AFM measurements. (E) AFM mapping experiments of starved, insulin, purslane, ginger, and tindora treated cells, respectively. Force curves were acquired on a surface area of 250 nm x 250 nm with a step size of approximately 7–8 nm. White pixels represent positive unbinding events, whereas black pixels depict no binding.

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