GM1-deficient C6 cells are sensitized to CT by inhibition of sialylation or GSL biosynthesis.

(A-E) C6 cells were cultured with the indicated inhibitors for 72 h followed by: (A) Staining was then performed with biotin-CTB, followed by DTAF-streptavidin. Fluorescence was measured by flow cytometry, represented here by MFI. (B) 1 h exposure to CT after which accumulated cAMP was measured by the cAMP-Glo™ luminescence assay. Luminescence signal is inversely proportional to cAMP levels. (C) As in panel A, but stained with biotin-PNA, followed by DTAF-streptavidin (D) Cell lysates were separated by PAGE and probed with biotin-PNA, biotin-CTB, or no biotinylated reagent, followed by streptavidin-peroxidase conjugate and development with chemiluminescent substrate. Equivalent amounts of protein were loaded in each lane and blots were probed with an anti-α-tubulin or anti-GAPDH antibody to confirm equivalent loading. (E) As in panel B, but brefeldin A (BFA) was added 1 h prior to CT addition and was also present during CT induction.