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Figure 1 | BMC Cell Biology

Figure 1

From: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

Figure 1

Increased integrin α5 expression correlates with enhanced bacterial adherence and uptake in vinculin -/- cells. (A) Vinculin WT and vinculin -/- cells were seeded on fibronectin coated glass bottom imaging dishes, next day fixed and stained for vinculin expression using α-human vinculin antibody (clone hVIN1). Total amounts of integrins were stained using integrin β1 antibody (clone HMβ1-1). TIRF microscopy was used to assess the localization of vinculin and β1 integrins. Bars represent 10 μm. (B) Equal amounts of protein in the whole cell lysates (WCL) of vinculin WT and vinculin -/- cells were separated by SDS-PAGE and processed for Western blotting using antibodies against talin, FAK, vinculin, paxillin, Src, or β-tubulin as indicated. Representative blots are shown. (C and D) Cells were seeded on gelatine coated 24 well plates and infected with S. aureus or S. carnosus with MOI 20 for 2 h. Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (C) or gentamicin protection assays (D), respectively. Values are means ± SEM from 5 independent experiments done in quadruplicate (n=20). Statistical significance of data was assessed by Mann-Whitney test. (E) Cells were stained for surface expression of β1, α5, αv, or β3 integrins and examined by flow cytometry. Data are shown as mean fluorescence intensity (MFI) and are corrected for the background MFI of integrin β1 -/- fibroblasts (β1 integrin staining) or corrected for the MFI of cells stained in the absence of primary antibody (α5, αv, and β3 integrin stainings). Results are shown as mean ± SEM from 3 independent experiments. (F) mRNA levels of β1, α5, αv, or β3 integrin transcripts in vinculin WT or vinculin -/- cells were analyzed by qRT-PCR. Integrin expression was normalized to GAPDH expression and was set to 1 for vinculin WT cells. Bars represent the mean of two independent experiments each performed in triplicate.

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