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

Figure 2

Validation of NKCC1 antibodies in COS7 cells. (a) Representative immunoblot of protein lysates obtained from COS7 cells and developed by using T4 or ckNKCC1. Shown is NKCC1 protein expression detected as two major bands, one of ~130 kDa and another of ~170 kDa. ((b)-(c)) Representative immunoblots of liver (b) or kidney (c) tissue extracts from WT or mice, developed by using T4 (b) or ckNKCC1 (c). Shown are bands of predicted MWs corresponding to NKCC1 only in WT tissues. (d) Representative immunoblots of and protein extracts developed by using T4. Shown are bands of predicted MWs corresponding to NKCC1 only in cells. ((e)–(h)) Representative images of cells stably expressing shRNAs against NKCC1 and GFP as a reporter (e) immunolabeled by using T4 coupled to Cy3-conjugated secondary antibodies (f). Cell nuclei were counterstained with DAPI (g) and the pictures superimposed (h). ((i)-(j)) Immunofluorescence of COS7 cells using T4 (i) or ckNKCC1 (j) coupled to FITC-conjugated secondary antibodies. Arrowheads indicate NKCC1-immunoreactivity at/near the edge of cells. Pictures in (e)–(h) and (i)-(j) were taken at 400x and 600x, respectively. Bar represents 10 μm.

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