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. Author manuscript; available in PMC: 2018 Jul 18.
Published in final edited form as: Biochemistry. 2017 Jul 3;56(28):3669–3681. doi: 10.1021/acs.biochem.7b00359

Figure 7. Flow cytometric analysis of surface expression of WT and mutant α11.2 co-expressed with β2a and α2δ1.

Figure 7

Surface HA expression of HA- and CFP-tagged WT and mutant α11.2 subunits in intact (PI-negative) HEK293T/17 cells assessed by flow cytometry. A) Representative histogram overlays (see key) of Alexa-fluor 647 signals from total PI-negative populations of anti-HA immunostained mock-transfected HEK293T/17 cells (black dotted line), no-antibody (non-stained) control (grey-shaded) and immunostained (red line) WT α11.2 samples and immunostained α11.2 mutants Q1655A (blue line), I1654A (brown line), Y1649A (gold line) and K1647A (green line). B) Histogram overlays of Alexa-fluor 647 signals from CFP-positive, PI-negative populations of no-antibody (non-stained) control (grey-shaded) and anti-HA immunostained (red line) WT α11.2 samples and immunostained α11.2 mutant Q1655A (blue line), I1654A (brown line), Y1649A (gold line) and K1647A (green line) samples from the same experiment shown in A. C) The amount of surface expressed (anti-HA stained) α11.2 (Alexa-647 fluorescence) relative to total channel (CFP signal intensity) in the transfected cell population is depicted in 2D contour plots. The upper-right plot shows contour plot of immunostained CFP-vector alone transfected control sample used to set the limit for the anti-HA signal. The upper left plot depicts anti-HA immunosignal on CFP-positive cell population of WT-α11.2 expressing cells. Unstained WT-α11.2 sample showed a similar fluorescence profile as the anti-HA antibody-stained CFP control (data not shown). The surface HA-immunosignal exhibited by the indicated α11.2 channel mutants from the same experiment is shown in 2D contour plots directly below. D) Bar graph of experimental results from repeat flow cytometry experiments. Anti-HA positive events as percent of total CFP positive events for each of the mutant CaV1.2 expressors normalized to the percent HA-positive observed for CFP positive population of WT CaV1.2 test samples in the same experiments (**= p<0.01 and ***= p<0.001, NS=p>0.05, Tukey post hoc tests; N=5–7). E) Representative immunoblot (left) and quantitative Bar graph analysis (right) of total α11.2 expression in samples used for flow analysis of α11.2 surface expression (p>0.05, N=5–7).