Fig. 3
Depleting all gangliosides in Sial9/Galgt1 double-KO neurons reduced binding and entry of BoNT/DC. a–c Binding of BoNT/C1 (a), BoNT/D-HC (b), and BoNT/DC-HC (c) to WT, Sial9/Galgt1 double-KO, and Sial9/Galgt1 double-KO neurons pre-loaded with exogenous gangliosides were examined by immunofluorescence staining, under the same assay conditions described in Fig. 1b–d. Binding of BoNT/C1 and BoNT/D-HC was abolished in Sial9/Galgt1 double-KO neurons. Binding of BoNT/DC-HC was reduced in Sial9/Galgt1 double-KO neurons. Exogenous gangliosides restored binding of all three toxins to Sial9/Galgt1 double-KO neurons. d–f WT and Sial9/Galgt1 double-KO neurons were exposed to BoNT/C1 (d), BoNT/D (e), or BoNT/DC (f) at the indicated concentrations. Cell lysates were analyzed by immunoblot as described in Fig. 1e–g. Entry of BoNT/C1 and BoNT/D into Sial9/Galgt1 double-KO neurons was largely blocked, as there was a lack of cleavage of SNAP-25 and syntaxin 1 (d), and VAMP2 (e). BoNT/DC still entered Sial9/Galgt1 double-KO neurons and cleaved VAMP2, but the levels of VAMP2 cleavage were lower in Sial9/Galgt1 double-KO neurons than in WT neurons (f), indicating that entry of BoNT/DC was reduced in Sial9/Galgt1 double-KO neurons compared to WT neurons. g–i Pre-loading exogenous gangliosides onto Sial9/Galgt1 double-KO neurons restored functional entry of BoNT/C1 (10 nM, g), BoNT/D (100 pM, h), and BoNT/DC (100 pM, i), analyzed by immunoblot as described in Fig. 1e–g. One of two (a–c) or three (d–i) independent experiments is shown
