Accessibility of primary amines and Cys in HsPCFT wild-type, Cys-, and single-Cys constructs.

<p>(<b>A</b>) Fifteen residues were selected within and close to ECL1 and ECL2. (<b>B</b>) Six residues were investigated in each ECL3 and ECL4. (<b>C</b>) Seven residues each were probed in ECL5 and ECL6. NH2-biotinylation shows Western blot after plasma membrane fraction isolation following surface-NH₂ biotinylation with sulfo-NHS-LC-biotin / avidin beads. Uninjected oocytes (uninj.) served as negative control, and PCFT V5 and PCFT CYS- injected oocytes served as positive control. SH-biotinylation shows Western blot after avidin isolation of MTSEA-biotin labeled Cys. Single-Cys engineered at positions indicated on top of the lanes. Equal amounts of protein samples were electrophoretically separated on 4-15 % precast gels, transferred to PVDF membranes, and probed with V5-HRP antibody (1/5,000 dilution in 5 % milk). The experiment was repeated on three different batches of oocytes and a representative Western blot is shown for each condition. Lanes in boxes are from separate experiments as additional positions were probed later.