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

Figure 3

NKCC1 is expressed in COS7 cells as a core/high-mannose, hybrid- and complex-type N-glycosylated protein. ((a)-(b)) Representative immunoblots of protein extracts from COS7 cells grown under basal conditions. Untreated protein extracts (control) or enzymatically digested with PNGaseF (a) or EndoH (b) were immunoblotted and developed using ckNKCC1 coupled to HRP-conjugated secondary antibodies. As loading control, membranes were probed with antibodies against β-actin. (c) Densitometry analysis of immunoblots of proteins control or treated with PNGaseF or EndoH. Shown are the estimated levels of the two main bands of NKCC1, that is, ~170 kDa (red bars) and ~130 kDa (green bars), before and after enzymatic deglycosylation. Data are expressed as means ± SEM (). (d) Pie-chart representing the approximate relative contribution of core/high-mannose, hybrid- and complex-type N-glycans to total endogenous NKCC1 in COS7 cells. Results are calculated based on the percent of increase in the densitometry signal of bands of ~130 kDa after enzymatic treatment.

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