A: Confocal images of Alexa-labeled C16-GM1 isoforms (3.2 μM C16:1-GM1, or 4.0 μM C16:0-GM1) in MEF-GM1−/− cells after 5 hour incubation with 40 nM CTB-A647. Inverted grayscale images of Sec61a-EGFP for the entire cell (far Left, bar = 10μm), 3-color merge and grayscale images of the individual channels (right panels, bar = 10μm).
B: Fraction of MEF-GM1−/− cells showing Sec61/ER colocalization for Alexa-labeled C16:1 and C16:0 following CTB treatment for 5 hours. n = 112 and 83 cells counted respectively.
C: Experiments performed as in (B) but with no CTB crosslinking.
D: Doses probed for each unlabeled GM1 isoform incorporated into the PM of MEF-GM1−/− cells as measured by enzymatic assay for surface-bound CTB-HRP conjugate. Cells were incubated with 20 nM CTB-HRP to label the PM at 4°C. Filled-bars indicate the conditions used in subsequent studies to load cells equally (red dotted line). (mean ± SEM, N = 3)
E: Fraction of MEF-GM1−/− cells showing Sec61/ER colocalization for unlabeled C16-GM1 variants following 20 nM CTB-Alexa594 treatment for 5 hours.
F: Time course of toxin-induced intracellular cAMP production in MEF-GM1−/− cells. (mean ± SEM, N = 3)
Bar graphs in B, C & E show mean ± SEM for N = 3 independent scores.
See also Figure S4.