Fig. 4.

VHH ALc-B8 and BLc-B10 specifically bind to their target Lc expressed in neuronal cell extracts. Neuro2a cells were co-transfected with expression plasmids either for ALc-B8 or BLc-B10 and for BoNT/A-Lc or BoNT/B-Lc as indicated. The Lcs were expressed as fusions to GFP and the VHHs were fused to YFP and a streptavidin binding peptide (see Section 2). After 24 h post-transfection, cell extracts were prepared and the ALc-B8 or BLc-B10 VHHs were incubated with streptavidin beads. The bound or unbound fractions following streptavidin affinity purification were obtained and the samples were resolved by SDS-PAGE and a Western blot was performed with anti-GFP antibody (Santa Cruz) that recognizes both GFP and YFP.