Volume 6 issue 6 June 2005
The Listeria protein InIB mimics Hepatocyte Growth Factor-induced Receptor trafficking
Ning Li, Guang-Sheng Xiang, Hatem Dokainish, Keith Ireton and Lisa A. Elferink

SUPLEMENTARY MATERIAL:

 Figure S1: Clathrin-dependent internalization of InlB in Vero cells. Internalization of Alexa-InlB into Vero cells transiently expressing dominant negative Eps15-EH29 or control, untransfected cells was quantified by confocal microscopy and is expressed as the mean fluorescence intensity of 20 or more randomly selected cells ± S.E.

Supplementary figure S1 (.tif)
Supplementary figure S1 (.gif)

 Figure S2: Arf6-GTP expression induces formation of cell protrusions. Hela cells transiently expressing HA-tagged, GDP-bound Arf6T27N or the constitutively active mutant Arf6Q67L were costained with FITC-labeled phalloidin to visualize actin filaments and anti-HA antibodies (Red) before processing for confocal microscopy. Membrane ruffles enriched in actin filaments are indicated (arrows). Bar, 10µm.

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Figure S3. Wortmannin disrupts the binding of EEA1 and Hrs to early endosomal membranes. T47D/cMet cells transiently expressing GFP-EEA1 or GFP-Hrs were incubated cells in the absence (-) or presence (+) of 100 nM Wortmannin for 1 hr at 37°C. The cells were either fixed directly (GFP-EEA1) or permeablized with 0.05% saponin for 30 min at 37°C to enable removal of soluble protein prior to fixation (GFP-Hrs) and examined using confocal microscopy. Bar, 10µm.

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 Figure S4.  Dominant negative PI3K inhibits InlB-induced membrane ruffling. Control (Con), untransfected T47D/cMet cells and cells transiently expressing dominant negative HA-DΔp85 were incubated in the absence (-) or presence (+) of 100 ng/ml InlB for 5 min at 37°C.  The cells were fixed and stained with anti-HA antibodies to detect cells expressing HA-ΔD p85 (Red) and costained with FITC-labeled phalloidin to visualize actin filaments, and examined using confocal microscopy. Membrane ruffles enriched in actin filaments are indicated (arrows). Bar, 10 µm.

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 Figure S5: Internalized Alexa-InIB colocalizes with endosomal Hrs. T47D/cMet cells transiently expressing GFP-Hrs (Hrs) were allowed to internalize Alexa ⁵⁹⁴-labeled InIB (InIB) for 15 min before processing for confocal microscopy. Areas of colocalization in the merged image appear yellow (arrowheads). Bar, 10µm.

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