Supporting Information
Mahdavi et al. 10.1073/pnas.1301740111
Movie S1.
Proteomic labeling of internalized
Yersinia
inside infected HeLa cells in the presence of gentamicin.
Y. enterocolitica
(T3SS-Wt) were diluted 1:25
from overnight cultures in LB and incubated at 26 °C with agitation until an OD
600
=
0.5 was reached. Infection was initiated at a multiplicity of infection of 100
in Opti-MEM. After 1 h of infection, 80
μ
g/mL gentamicin was added. After 1 h, the medium was changed to fresh Opti-MEM, and 1 mM Anl was added to both
samples. The medium was supplemented with 4
μ
g/mL gentamicin to maintain inhibition of protein synthesis by extracellular bacteria. After 3 h of labeling,
cells were fixed with 3.7% formaldehyde for fluorescence confocal microscopy. Alexa Fluor 633 conjugated to wheat germ agglutinin (WGA) was used to l
abel
the membranes of HeLa cells; the associated fluorescence is shown in red. Anl-labeled proteins were treated with alkyne-functionalized Alexa Fluor
488; the
associated fluorescence is shown in green. Confocal fluorescence microscopy was used to image a series of Z-sections through the infected HeLa cells
. Z-stacks
were taken from the top of the HeLa cells to the bottom of the glass surface. Internalized
Yersinia
cells are labeled inside infected HeLa cells. Extracellular
bacteria are not labeled, because protein synthesis is inhibited in these cells.
Movie S1
Mahdavi et al.
www.pnas.org/cgi/content/short/1301740111
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Movie S2.
Proteomic labeling of internalized and extracellular
Yersinia
in HeLa cell infections lacking gentamicin. Infections were performed under identical
conditions as in
Movie S1
but lacking gentamicin. Labeling times were identical to those for
Movie S1
. Alexa Fluor 633 conjugated to WGA was used to label
the membranes of HeLa cells; the associated fluorescence is shown in red. Anl-labeled proteins were treated with alkyne-functionalized Alexa Fluor
488; the
associated fluorescence is shown in green. Confocal fluorescence microscopy was used to image a series of Z-sections through the infected HeLa cells
. Z-stacks
are taken from the top of the HeLa cells to the bottom of the glass surface, showing labeled bacteria on the surface of the glass substrate. Anl was in-
corporated during protein synthesis by the internalized and extracellular
Y. enterocolitica
, and both populations are labeled in green.
Movie S2
Other Supporting Information Files
SI Appendix (PDF)
Mahdavi et al.
www.pnas.org/cgi/content/short/1301740111
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