FIG 2.

Enhancement of bacterial invasion by IgG, fibrinogen, fibronectin, and fetal bovine serum. (A) The effects of matrix proteins laminin (LMN), IgG, fibrinogen (Fg), and fibronectin (Fn), and of fetal bovine serum (FBS), on the invasion of HaCaT cells by S. aureus were investigated. A total of 10⁶ HaCaT cells were stimulated either with 10% FBS or with 10 μg/ml of IgG, Fg, or Fn and were then infected with USA300 or its Δlpl mutant at an MOI of 30. The controls (C) were infected with bacteria only. The host cells were infected for 1.5 h, followed by lysostaphin treatment for another 1.5 h to kill adherent S. aureus cells. All experiments were performed at least in triplicate in three independent replications. Error bars indicate standard deviations. Statistical significance was calculated by using 2-way ANOVA (ns, no statistical difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (B) Far-Western blotting was performed with different matrix proteins to determine the possible binding partners. A polyacrylamide gel was loaded with 10 μg of BSA, Fg, Fn, IgG, LMN, PLG, or Vn. Proteins were blotted onto a nitrocellulose blotting membrane and were subsequently probed with 100 μg Lpl1. For immunoblotting, an anti-Lpl1 antibody was used as the first antibody, and goat-anti-rabbit IgG was used as the secondary antibody. Detection was performed with a BCIP/NBT solution.