RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization (original) (raw)
Figure 4
Rac1 and Cdc42 are required for invasion of P. aeruginosa into mammalian cells.
(A) HeLa cells were treated with Rac1, Cdc42 or control (c) siRNA. Cell lysates were immunoblotted with anti-Rac1 and anti-Cdc42, respectively. GAPDH was used as loading control. (B) Internalization of PAK, PAKΔS, PAKΔT and PAKΔSΔT at 1 h was quantified in HeLa cells treated with control siRNA or siRNA against Rac1 or Cdc42. *p<0.05, **p<0.01 compared to control RNA-treated cells for each bacterial strain. (C–F) GTPase activation assays. HeLa cells were infected with PAK, PAKΔSΔT, PAKΔS and PAKΔT for the indicated times. Lysates were incubated with Pak1-PBD to precipitate GTP-bound Cdc42 and GTP-bound Rac1. The bound proteins and cell lysates were examined by immunoblotting with anti-Cdc42 (C) and anti-Rac1 (E), respectively. The experiments were performed 3 times and a typical gel is shown. (D, F) The GTPases activation assays to assess Cdc42 (D) or Rac1 (F) activation upon infection with PAK, PAKΔSΔT, PAKΔS and PAKΔT were quantified by densitometry. Shown are the mean values +/- SD from three independent experiments.