Accessibility of primary amines and Cys in HsPCFT wild-type, Cys-, and single-Cys constructs.
(A) Fifteen residues were selected within and close to ECL1 and ECL2. (B) Six residues were investigated in each ECL3 and ECL4. (C) 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.