Fig. 6. The cysteine-rich domain of SNAP-25 is important for SNARE complex formation in intact cells. (A) Post-nuclear supernatants in sample buffer were prepared as described in the Materials and Methods. The samples were either not boiled (-) or boiled (+) and loaded on a 9% SDS-PAGE gel. SNAP-25 complexes from G14 cells transiently transfected with 5 µg of POMC-ß-Gal together with 5 µg of pcB7 or SNAP-25A/ER-pcB7 (S25A) or CA-SNAP-25A/ER-pcB7 (CA S25A) were detected by western blot analysis with anti-SNAP-25 antibody. The arrowhead indicates SNARE complexes formed by exogenous SNAP-25 and the double arrow indicated SNAP-25 complexes formed by endogenous SNAP-25. (B) The western blot of a similar experiment was probed with anti-Myc antibody. (C) The total amount of SNAP-25 protein expressed in the transfected cells shown in A is detected with the anti-SNAP25 antibody. (D) SNAP-25 complexes from G14 cells transiently transfected with POMC-ß-Gal together with either SNAP-25A/ER-pcB7 or Delta-SNAP-25A/ER-pcB7 were detected as described in A. (E) The total amount of SNAP-25 monomer expressed in these cells was detected as in C. These experiments were repeated three times with triplicate samples per experiment. (F) SNAP-25 complexes from G14 cells transiently transfected with the indicated amounts (µg) of POMC-ß-Gal and CA-SNAP-25A/ER-pcB7 or CA-SNAP-25A/ER-pcB7 alone. The arrowhead indicates SNARE complexes formed by exogenous SNAP-25 and the double arrow indicates complexes formed by endogenous SNAP-25. The total amount of CA-SNAP-25A/ER expressed in the cells is shown in the lower panel. The western blot was probed with the anti-SNAP-25 antibody.

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