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. 2016 Dec 5;26(12):1284–1296. doi: 10.1093/glycob/cww075

Fig. 2.

Fig. 2.

Overexpression of GPC4 enhances the IIH6 immunoreactivity of the LARGE2-expressing DG−/− cells. (A) Schematic representation of the myc-tagged GPC4 (Myc-GPC4) construct. The suggested proteolytic cleavage site and potential GAG attachment sites are indicated by an arrow and vertical lines, respectively. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. The DG−/− cells stably expressing LARGE2 were transfected with Myc-GPC4. (B) Clones stably expressing myc (#4, #14 and #22) were analyzed by flow cytometry for cell-surface staining with IIH6 or anti-myc antibody. (C) Lysates of these cells enriched for Myc-GPC4 by DEAE-enrichment were immunoblotted with IIH6 or anti-myc antibody. Note that the enhanced intensity of the IIH6 staining is correlated with that of anti-myc staining. (D) Representative immunofluorescence micrograph of the DG−/− cells stably co-expressing LARGE2 and Myc-GPC4 (clone #4). The cell surface was stained with IIH6 and anti-myc antibody in the absence of permeabilization. Scale bar, 50 μm. The top image shows IIH6 immunofluorescence, the middle image shows myc immunofluorescence, and the bottom image shows IIH6 (red) merged with Myc (green) and DAPI (blue). All myc staining colocalizes with IIH6 as no ‘green only’ fluorescence is seen. This figure is available in black and white in print and in color at Glycobiology online.