Journal: Cell reports
Article Title: Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread
Figure Lengend Snippet: IpaC Interactions with β-Catenin Are Associated with S. flexneri Intercellular Spread (A) Schematic of yeast protein-protein interaction platform. Interaction of the mCherry-tagged prey protein (red) with the bait protein fused to the inclusion body forming protein μNS (gray) results in puncta of red fluorescence. In contrast, the lack of an interaction between prey and bait proteins results in generally diffuse mCherry fluorescence throughout the cytosol of the yeast cells. (B) Protein interaction assay. Arrowheads, fluorescent puncta. Representative images. A total of 88–186 yeast were analyzed per condition per experiment. Scale bar, 10 μM. (C) Percentage of yeast displaying puncta, which indicates an interaction; mean ± SEM. (D and E) Infection of HeLa cells with or without β-catenin knockdown by S. flexneri Δ ipaC -producing WT IpaC or IpaC R362W. (D) Bacterial plaques formed in monolayers stably expressing β-catenin-targeting (#1 and #2) or control shRNA. Images collected at 18 h of infection. Green, S. flexneri ; blue, DNA. Scale bar, 100 μm. Representative images. Five to ten fields were examined per condition per experiment. (E) Quantification of plaque size (area of spread) from (D), mean ± SEM. Dots represent data from three or more independent experiments (C and E). *p
Article Snippet: Western BlotsWestern blots were performed using 1:10,000 mouse anti-β-catenin (BD Biosciences, 610153), 1:1000 mouse anti-vinculin (Sigma, SAB4200729), or 1:1000 rat anti-N-cadherin [Developmental Studies Hybridoma Bank, MNCD2, ( )], 1:1000 rat anti-E-cadherin (Thermo, 14-3249-82) incubating overnight at 4°C, or 1:500 rabbit anti-α-catenin (Thermo, 71-1200), 1:1000 rabbit anti-caveolin 1 (Sigma, C4490), 1:1,000,000 rabbit anti-GroEL (Sigma, G6532), 1:1000 mouse anti-GAPDH (Developmental Studies Hybridoma Bank, DSHB-hGAPDH-2G7), 1:40,000 rabbit anti-keratin 8 (Abcam, Ab53280), 1:10,000 rabbit anti-keratin 18 (Abcam, ab668) 1:5000 goat anti-rabbit HRP (JacksonImmuno, 111-035-144), 1:5000 goat anti-rat HRP (JacksonImmuno, 112-035-003), and 1:5000 goat anti-mouse HRP (JacksonImmuno, 115035003) for 2 hours at room temperature.
Techniques: Fluorescence, Protein Interaction Assay, Infection, Stable Transfection, Expressing, shRNA