Extended Data Fig. 5: Validation of GSLs as decoy receptors for CT.
From: Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication
(A) Lectin blot with CTB-biotin of control and B3GALT5-KO m1 cell lysates, and pure GM1. Samples were treated for 16 h with endoglycoceramidase or a vehicle control. Data shown are a single representative trial of 3 independent biological replicates. (B-G) Control, B3GNT5-KO + OE, and B3GALT5-KO m1 cells were treated with P4 inhibitor of glycosphingolipid biosynthesis for 72 h then lysed for lectin blot analysis (B) or treated with BFA or a vehicle control for 0.5 h prior to incubation with CT (1 nM) for analysis of cAMP accumulation (C, D, and E). Alternately, cells were treated with forskolin (10 µM) for 0.5 h (F and G). Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate (n = 2 in panel E, n = 3 in panels C, D, F, and G) consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. Statistical analyses were performed by two-way ANOVA with Tukey correction. Exact P-values are 0.0002 for control versus inhibitor-treated B3GALT5-KO m1 as well as inhibitor-treated control versus untreated B3GALT5-KO m1; for inhibitor-treated control versus inhibitor-treated B3GALT5-KO m1, the exact P-value is 0.0052 (panel C).
