Journal of Signal Transduction / 2015 / Article / Fig 5

Research Article

Analysis of AKAP7 Dimerization

Figure 5

Photon counting histogram detects dimerization of AKAP7γ in live cells. (a) A representative 0.5 s trace is shown to demonstrate the fluctuation in intensity over time as particles enter and leave the measurement volume (A). The full 10 s fluctuation trace is divided into 10 μs bins and plotted as a histogram of frequency versus counts/bin (B). Fitting of the histogram yields the concentration (number, ) and brightness (ε) of the fluorescent particles. (b) Data summary. HEK-293 cells were transfected with monomeric EGFP, dimeric EGFP, or AKAP7γ-EGFP. Selected cells were measured 5 times at 5 different positions within the cytosol for 10 s per measurement. Measurements exhibiting bleaching or other instabilities in fluorescence intensity were excluded from analysis. Global fitting of the data produced near ideal fits ( of 1.0 is ideal). Monomeric and dimeric EGFP were fit using a single-component model, while AKAP7γ was fit using a 2-component model allowing for the detection of both monomeric and dimeric AKAP7γ-EGFP. Average concentrations and [dimer]/[total] were determined from the 52 measurements in which the presence of dimer was detected. (c) Known quantities of purified AKAP7γ-S-tag were subjected to SDS-PAGE alongside lysate obtained from 250,000 human aortic smooth muscle cells. Using the standard curve created from densitometry measurements of the known quantities of AKAP7γ-S-Tag, we were able to estimate the amount of AKAP7γ per cell. The mass of AKAP7γ per cell was divided by its molecular weight (37 kD) to convert mass to moles and then divided by a cellular volume of 30 pL to arrive at a final cellular concentration of 180 nM. This volume is the approximate volume of cardiac myocytes, which we expect to be similar to that of an aortic smooth muscle cell [26].
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