Supporting Online Material


Ankyrin Repeat Proteins Comprise a Diverse Family of Bacterial Type IV Effectors
Xiaoxiao Pan, Anja L�hrmann, Ayano Satoh, Michelle A. Laskowski-Arce, Craig R. Roy

Supporting Online Material

This supplement contains:
Materials and Methods
Fig. S1
Tables S1 and S2
References

This file is in Adobe Acrobat PDF format.

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/320/5883/1651/DC1)
Movies S1 to S3

Movie s1
Golgi fragmentation mediated by Brefeldin A. BSC-1 cells that stably produce GalNAc-T2-YFP were pretreated with cycloheximide (10 μg/ml) and Brefeldin A (5μg/ml). Cells were moved to a stage pre-warmed to 37° C and time-lapse confocal images were acquired every 30sec. Movie is displayed at 2 frames/sec and a timestamp is located in the lower right corner.

Movie s2
Golgi fragmentation mediated by AnkX. BSC-1 cells that stably produce GalNAc-T2- YFP were pretreated with cycloheximide (10 μg/ml). Cells marked with an asterix were injected with purified His-AnkX (1mg/ml) mixed with fluorescent dextran (injection marker). Image acquisition was initiated roughly 4min after injection as cells were moved from the injection microscope to the confocal microscope having a stage pre-warmed to 37° C. Confocal images were acquired every 30sec using an LSM510 microscope. Note that cells marked with an "M" are dividing in the movie and the Golgi can be observed fragmenting and then reassembling, indicating that the fragmentation induced by AnkX was not reversible in this period of time. Movie is displayed at 2 frames/sec and a timestamp is located in the lower right corner.

Movie s3
Defects in transferrin sorting mediated by AnkX. CHO-T cells were treated with NaOBt (5mM) for 16h to induce the expression of the human transferrin receptor. Before injection, cells were washed and incubated with serum-free tissue culture medium for 45 min. Cells were injected with purified His-AnkX (1mg/ml) mixed with fluorescent dextran (injection marker) and placed on ice for 5 min. Alexa488-Tf diluted 1:100 was added to cells on ice for 45 min. Cells were washed with cold medium to remove unbound transferrin. Image acquisition was initiated roughly 4min after removal from ice as the cells were moved to a stage prewarmed to 37° C. Confocal images were acquired manually for 4min then automatically every 30sec. The last frame of this movie is shown in Figure 3D and the arrow indicates the location of the injected cell. Movie is displayed at 2 frames/sec and a timestamp is located in the lower right corner.

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