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. 2000 Oct 16;151(2):209–220. doi: 10.1083/jcb.151.2.209

Figure 1.

Figure 1

Expression of AP-tagged EGFR ligands. (a) Structure of the AP-tag parental expression plasmid, pSS-AlPh. (b) A schematic diagram of the region within the expression plasmids derived from pss-AlPh that encode the fusions of AP-tag and EGFR ligands. The coding region for the AP-tag HB-EGF fusion was constructed such that, in the encoded fusion, the AP sequence (488 amino acids) was inserted at Ala84 of the HB-EGF precursor. In the AP-tag AR and AP-tag TGF-α fusion proteins, AR (residues 102–252) and TGF-α (residues 45–160), respectively, were substituted for HB-EGF (residues 85–208). (c) Expression of AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α on the surface of transfected CHO cells. Biotinylated AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α were immunoprecipitated with human placental AP antibody and analyzed by SDS-PAGE and Western blotting. Biotinylated proteins on the Western blot membranes were detected by HRP-conjugated avidin and ECL. (d) AP activity in the conditioned media of transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α. Cells were treated with or without 60 nM TPA for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values.