Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride Cotransporter Slc12a2

Figure 5

NKCC1 localizes in the ER of COS7 cells. ((a)-(b)) Representative image of COS7 cells immunolabeled against NKCC1 using T4 (a) or calreticulin (CRT, (b)) antibodies and developed using fluorescently labeled secondary antibodies: Cy3 (NKCC1, red) and FITC (CRT, green). (c) Overlay of (a) and (b). Pictures were superimposed to obtain an image where colocalization could be digitally estimated as yellow pixels (red + green = yellow). Scale bar represents 10 μm. (d) Semiquantitation of red, green, and yellow pixels corresponding to NKCC1, CRT, and NKCC1 + CRT, respectively. Shown are the results obtained from at least 10 cells and represented as mean fluorescence intensity in arbitrary units ± SEM. (e) Colocalization heat-map of the squared cell in (c) computed by using NIH ImageJ. ((f)-(g)) Shown are representative images of COS7 cells grown in the presence of cycloheximide (1 μg/mL) to acutely inhibit protein synthesis and deplete endogenous NKCC1 from the ER. The presence of NKCC1 (f) or CRT (g) was codetected by using the relevant primary antibodies and developed using secondary antibodies labeled with Cy3 (red) or FITC (green) fluorophores. (h) Superimposition of (f) and (g) images to estimate colocalization as heat-maps, as shown in (i).

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