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Fig. 1. Identification of SDS-resistant SNARE complexes in PC12 cell membrane extracts. SDS-membrane extracts were separated on SDS-PAGE, transferred to nitrocellulose membranes and were immunoblotted with polyclonal and monoclonal antibodies against SNARE. (A) Immunoblot of unboiled samples and samples that had been boiled before electrophoresis from a single membrane preparation using MAB333 ( ${alpha}$ SB¹), MAB331 ( ${alpha}$ S25) and MAB336 ( ${alpha}$ STX¹). The insert at the bottom shows the anti-SNARE bands that correspond to the monomeric antigens from a shorter film-exposure of the nitrocellulose membrane. Of the three antibodies only MAB331 (antibody against SNAP-25) detects heat-sensitive SDS-resistant SNARE complexes. However, the amount of monomeric syntaxin and synaptobrevin is increased in boiled samples, suggesting that in the unboiled samples a fraction of these proteins is engaged in protein complexes where epitopes are inaccessible for the respective antibody. Immunoblots shown in B to E were performed with unboiled SDS-samples. (B) Syntaxin antibody clone HPC-1 ( ${alpha}$ STX²) detects, besides the monomeric antigen at about 36 kDa, two high-molecular weight bands at positions, which correspond to the complex bands identified with MAB331 and shown in A (at $~$ 230 and $~$ 100 kDa). (C) Syntaxin-antibody clone 78.3 ( ${alpha}$ STX³) recognizes only the 230 kDa protein band (with low staining intensity) which is also detected by MAB331 ( ${alpha}$ S25), whereas synaptobrevin antibody clone 69.1 ( ${alpha}$ SB²) fails to detect any slow migrating bands. (D) After heat-treatment of the nitrocellulose membrane, ${alpha}$ STX³ recognizes both high-molecular weight bands that are also detected by MAB331 ( ${alpha}$ S25). (E) On heat-treated nitrocellulose membranes, synaptobrevin antibody clone 69.1 ( ${alpha}$ SB²) detects the 230 kDa band, whereas synaptobrevin antibody AB5856 ( ${alpha}$ SB³) recognizes only the 100 kDa protein band. Note that the samples separated in lanes 1-3 and lanes 4 and 5 (from left to right), were from the same membrane preparation. The preparation analysed in lanes 4 and 5 contained a third SDS-resistant band at about 55 kDa (asterisk). The recognition patterns of the antibodies were verified in at least three independent experiments.

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