Figure 4.
GxTX-594 selectively labels Kv2 proteins.(A) Fluorescence from live CHO cells transfected with Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP + KChIP2, Kv1.5-GFP + Kvβ2, or BK-GFP (indicated by row) and labeled with GxTX-594. Airy disk imaging was from a plane above the glass-adhered surface. Cells were incubated with 100 nM GxTX-594 and 5 μg/ml WGA-405 then rinsed before imaging. Fluorescence corresponds to emission of GFP (column 1), GxTX-594 (column 2), WGA-405 (column 3), or an overlay (column 4). Scale bars, 20 μm. (B) Intensity of GxTX-594 labeling for different K+ channel-GFP types. Fluorescence intensity resulting from 594-nm excitation of GxTX-594 is divided by fluorescence intensity resulting from 488-nm excitation of GFP. This value was normalized to the average 594:488 ratio from GxTX-594 and Kv2.1-GFP. Circles indicate measurements from individual cells. Only cells with obvious GFP expression were analyzed. For analysis, ROIs were drawn around the cell membrane indicated by WGA-405 fluorescence. Pixel intensities were background subtracted before analyses by subtracting the average fluorescence of a background ROI that did not contain cells from the ROI containing the cell; this occasionally resulted in ROIs with negative intensity. Kv2.1, n = 16; Kv2.2, n = 10; Kv4.2, n = 13; Kv1.5, n = 13; and BK, n = 10; n indicates the number of individual cells analyzed in a single dish during a single application of GxTX-594 with the indicated K+ channel-GFP type. Bars represent the mean. Significant differences were observed between 594:488 ratio for Kv2.1 or Kv2.2 and Kv1.5, Kv4.2, or BK by Mann–Whitney U test (P < 0.0001). The P value to determine significance is adjusted for multiple comparisons using the Bonferroni method, where P < 0.0033 is considered significant, with the caveat that data points under each condition are technical replicates. (C) Pearson correlation coefficients between GxTX-594 and GFP. Same cells as B. Significant differences were observed between correlation coefficients for Kv2.1 or Kv2.2 and Kv1.5, Kv4.2, or BK by Mann-Whitney U test (P < 0.0001).

GxTX-594 selectively labels Kv2 proteins. (A) Fluorescence from live CHO cells transfected with Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP + KChIP2, Kv1.5-GFP + Kvβ2, or BK-GFP (indicated by row) and labeled with GxTX-594. Airy disk imaging was from a plane above the glass-adhered surface. Cells were incubated with 100 nM GxTX-594 and 5 μg/ml WGA-405 then rinsed before imaging. Fluorescence corresponds to emission of GFP (column 1), GxTX-594 (column 2), WGA-405 (column 3), or an overlay (column 4). Scale bars, 20 μm. (B) Intensity of GxTX-594 labeling for different K+ channel-GFP types. Fluorescence intensity resulting from 594-nm excitation of GxTX-594 is divided by fluorescence intensity resulting from 488-nm excitation of GFP. This value was normalized to the average 594:488 ratio from GxTX-594 and Kv2.1-GFP. Circles indicate measurements from individual cells. Only cells with obvious GFP expression were analyzed. For analysis, ROIs were drawn around the cell membrane indicated by WGA-405 fluorescence. Pixel intensities were background subtracted before analyses by subtracting the average fluorescence of a background ROI that did not contain cells from the ROI containing the cell; this occasionally resulted in ROIs with negative intensity. Kv2.1, n = 16; Kv2.2, n = 10; Kv4.2, n = 13; Kv1.5, n = 13; and BK, n = 10; n indicates the number of individual cells analyzed in a single dish during a single application of GxTX-594 with the indicated K+ channel-GFP type. Bars represent the mean. Significant differences were observed between 594:488 ratio for Kv2.1 or Kv2.2 and Kv1.5, Kv4.2, or BK by Mann–Whitney U test (P < 0.0001). The P value to determine significance is adjusted for multiple comparisons using the Bonferroni method, where P < 0.0033 is considered significant, with the caveat that data points under each condition are technical replicates. (C) Pearson correlation coefficients between GxTX-594 and GFP. Same cells as B. Significant differences were observed between correlation coefficients for Kv2.1 or Kv2.2 and Kv1.5, Kv4.2, or BK by Mann-Whitney U test (P < 0.0001).

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