Western blotting of Akt phosphorylation.

Hepatocytes were seeded (HepG2, 5 × 10⁵ cells/dish; Hep3B, PLC5, and Huh7, 3 × 10⁵ cells/dish) in 35-mm-diameter cell culture dishes and cultured for 48 h. For exposure to recombinant InlB, cells were washed and incubated for 30 min in serum-free medium and then incubated with or without 1.25 nM InlB for 5 min at 37°C. The cells were then washed with cold PBS and lysed with cold lysis buffer (150 mM NaCl, 20 mM Tris/HCl, 2 mM EDTA, 1% NP-40, 3 mM sodium orthovanadate, 50 mM sodium fluoride, and 1× EDTA-free protease inhibitor cocktail [Roche]). To assess the effect of InlB produced by L. monocytogenes, the cells were washed with medium without serum and infected with WT or InlB-deficient bacteria at an MOI of 20 for 30 min at 37°C (same experimental conditions as the invasion assay). The cells were then washed and lysed. Cell lysates were subjected to Western blot analysis using PVDF membranes and anti-Akt or anti-phospho-Akt (Ser473) antibodies (Cell Signaling) and secondary anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Cell Signaling).