Fig. 1. Shiga toxin B-subunit is transported via SNX1-positive endosomes to the TGN. Fluorescence-labeled Shiga toxin B-subunit (FITC-STxB, green channel) was surface-bound to HeLa cells at 4°C and subsequently allowed to internalize for the indicated times at 37°C or 19.5°C; cells were fixed and immunolabeled for endogenous SNX1 (Alexa594, red channel) and imaged using confocal microscopy. Maximum projections of eight to ten optical z-slices (480 nm z-separation) are shown. (A) Cells were fixed without incubation at 37°C (0 minutes). (B) After 10 minutes at 37°C, the Shiga toxin B-subunit extensively colocalized with the SNX1-positive endosomal structures (yellow color in merged images). Note the subset of FITC-STxB tubules negative for SNX1 (arrowheads in B and C). (C) Shiga toxin B-subunit accumulated in SNX1-positive endosomes when incubated at 19.5°C for 1 hour. When incubated for longer times at 37°C (D,E), it displayed a TGN-like enrichment at the juxtanuclear area. (F) At these time points, Shiga toxin B-subunit colocalised extensively with TGN46 (Alexa594, red). Bars, 10 µm.