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

Figure 8

The first step of N-glycan biosynthesis is required for NKCC1 protein expression. ((a)-(b)) Shown are representative immunofluorescence microscopy images of COS7 cells grown under control conditions (a) or treated for 16 h with 2 μg/mL tunicamycin (TUN, (b)). NKCC1 immunolocalization was analyzed with T4 and FITC-labeled secondary antibodies (green). (c) Representative immunoblot with ckNKCC1 demonstrating expression of NKCC1 in the biotinylated plasma membrane fraction purified from COS7 cells control or treated with TUN. Protein expression of cytosolic GAPDH was used to assess the purity of biotinylated plasma membrane fractions. (d) Densitometry scanning of the immunoblot in (c) representing the extent to which TUN (red trace) decreases plasma membrane located NKCC1 (black trace). (e) Representative immunoblot showing the expression pattern of total NKCC1 in COS7 cells in response to TUN 2 μg/mL during the indicated periods of time. As loading control, immunoblots were probed against tubulin.

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