Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen
Fig 4
Characterization of typhoid toxin trafficking to the TGN in CRISPR/Cas9-edited cell lines.
(A and B) Co-localization of typhoid toxin with the Golgi marker GM130. (A) Wild-type and the indicated knockout cell lines were treated with Oregon-488 labeled typhoid toxin (green) and 2 hours after toxin treatment, cells were stained with an anti-GM130 antibody (red) as described in Material and Methods. Scale bar, 5 μm. (B) The co-localization between typhoid toxin and GM130 was determined as described in Material and Methods. Values represent the relative co-localization (normalized to wild type) and are the mean ± SEM of three independent experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05; two-tailed Student’s t-test. (C and D) Typhoid toxin Golgi localization determined by SNAP-capture. (C) Cells expressing Myc-epitope tagged SNAP-GalT (Myc-SNAP-GalT) were incubated with BG-labeled typhoid toxin for 6 hr and subsequently analyzed by Western blot with an anti-Myc antibody to detect typhoid toxin/SNAP-GalT chimeric protein complexes (TT-SNAP-GalT) and anti β-actin antibody as a loading control. The migration position of the uncomplexed (Myc-SNAP-GalT) and toxin complexed GalT-SNAP (TT-SNAP-GalT) are indicated. (D) Relative amounts of TT-SNAP-GalT quantified from the blots as indicated in Materials and Methods. Values represent the relative intensity of all bands associated with TT-SNAP-GalT (normalized for loading and relative to the values of wild type, which were considered 100) and are the mean ± SEM of 3 independent determinations. ****p < 0.0001; ***p < 0.001; *p < 0.05; n. s.: differences not statistically significant; two-tailed Student’s t-test.