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. 2012 Jul 22;287(37):31104–31115. doi: 10.1074/jbc.M112.387498

FIGURE 2.

FIGURE 2.

LRP1-dependent VacA internalization and vacuolation in AZ-521 cells. A, confocal microscopic analysis of VacA binding to AZ-521 cells via LRP1. Nontargeting (NC) or LRP1 siRNA-transfected AZ-521 cells were incubated with Alexa 555-labeled VacA (red) for 30 min at 4 °C or for 1 h at 37 °C, fixed with 4% paraformaldehyde, and reacted with anti-LRP1 antibodies (green) as described under “Experimental Procedures.” The nuclei were stained with DAPI. A merged picture shows co-localization of VacA and LRP1 in AZ-521 cells. Bars represent 20 μm. Experiments were repeated two times with similar results. B, silencing of LRP1 gene inhibited VacA-induced vacuolation. The indicated siRNA-transfected AZ-521 cells were incubated with 120 nm heat-inactivated (IA) or wild-type VacA (A) for 18 h at 37 °C. Vacuolating activity was evaluated by neutral red uptake assay as described under “Experimental Procedures.” Data are presented as mean ± S.D. and significance is (*) p < 0.01 (n = 3). Experiments were repeated three times with similar results.