Fig. 1: Determination of mGFP–hNET oligomer distribution using single-molecule brightness analysis. | Communications Biology

Fig. 1: Determination of mGFP–hNET oligomer distribution using single-molecule brightness analysis.

From: Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux

Fig. 1

a Concept of TOCCSL runs in mGFP-hNET-transfected CHO cells. i) A prebleach image is recorded and an aperture-restricted area of the bottom cell membrane is subsequently photobleached for 2000 ms (tbl) with a laser intensity of ~2 kW/cm2 (Ibl). During 5,000 ms of recovery time, unbleached molecules re-enter the bleached area by Brownian motion; thereafter, a postbleach image is recorded in which individual molecules are distinguishable. The TOCCSL image was acquired with 5 ms excitation time (texp) and laser intensity of 0.4–0.6 kW/cm2 (Iim). The scale bar of the prebleach and postbleach image is 10 µm. ii) Timing protocol for a typical TOCCSL experiment, plotted as time vs. laser intensity. b Representative brightness distribution of the oligomeric fractions obtained from TOCCSL runs on ten cells, plotted as probability density function (PDF). c NET co-exists as monomers (~60%) and dimers (~40%) at the plasma membrane. Each data point represents an independent experiment with TOCCSL runs on 15 cells. Bars represent means ± SD.

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