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Figure 3
DEAE purification and identification of glypican-AP fusion proteins.  (A) Wash and eluted DEAE fractions of transfectant cell medium were separated on a 1% agarose gel and the protein bands were developed by incubation in an AP substrate overnight. Lanes 1 and 2 show 750 mM NaCl eluted fractions with high AP activity, diluted 1:2 and 1:10, respectively; lane 3 shows sample flow-through; lane 4 is a 150 mM NaCl wash; lane 5 is a 250 mM NaCl wash. (B) Cell media from cos-7 cell cultures, transfected with APtag-2 containing only coding sequence for AP (lane 1), coding sequence for neither AP nor glypican (lane 2), or coding sequences for both AP and glypican (lane 3), were heparitinase-treated, separated on a 12% SDS-PAGE gel, and probed with an anti-AP antibody.  The top band in lane 3 corresponds to 130 kd, the MW of the glypican-AP fusion protein.  The bottom bands correspond to 55 kd, the MW of AP.  The AP in sample 2 is believed to be endogenously produced.  (C) DEAE-purified fusion proteins were treated with heparitinase (lane 2), chondroitinase ABC (lane 3), or both (lane 4), separated on a 1% low-melting-point agarose gel and developed as in (A).  Lane 1 represents untreated protein sample.

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