Figure 4

Cell surface cross-linking reveals direct CasBrE Env and 4070A interactions in pseudotyping NSCs. A. Treatment of control, 4070A virus, or vector transduced C17.2 NSCs with the water soluble chemical cross-linker DTSSP to assess cell surface interactions between CasBrE SU or SU/TM and 4070A Env. Extracts from cross-linked cells were treated with or without dithiotheitol (DTT), separated by SDS-PAGE on 8% gels, followed by immunoblotting first for CasBrE Env (A-C, top panels; 697) followed by stripping and reprobing for 4070A viral proteins (A-C, lower panels; pig anti-AmLV). Cross-linked complexes (X-linked Env) appear at the top of the blots as slow mobility bands or smears, which were converted by DTT treatment to molecular mobility patterns consistent with un-crosslinked samples. B. Sequential immunoblotting of cross-linked samples from CasES+4070A-NSCs separated by non-reducing SDS-PAGE (without DTT) in the first dimension (horizontal) followed by reducing SDS-PAGE (with DTT) in the second dimension (vertical). The same sample was also treated with DTT and only run in the second dimension indicated by the separate lane (left columns) to mark the migration of the different immunoreactive components. C. Non-reducing/reducing two-dimensional SDS-PAGE analysis of crosslinked CasE+4070A NSC extracts. Arrows and arrowheads indicate the presence of the Env species that co-migrated in the high molecular weight cross-linked complexes.