Figure 6. Akt1 is the isoform regulating p40phox activation in AMs.

AMs were pretreated for 48 h with nontargeting control (CTR) or Akt1 (Akt1) siRNA (A–D) and then were stimulated with 50:1 IgG-K. pneumoniae (black) for 1 h (B) or 15 min (C and D). (B) The H₂O₂ concentration in medium was determined fluorometrically. (A, C, and D) Cells were lysed, and proteins were subjected to immunoblot analysis for (A) Akt1, (C and D) p-p40phox, or (D) p-ERK and p-PKCδ. (E) Cells were pretreated for 10 min with or without PGE₂ followed by the addition of 50:1 IgG-K. pneumoniae for 15 min. Cells were lysed, and p40phox was immunoprecipitated using a specific Ab. Then the p40phox immunoprecipitates were subjected to immunoblot analysis for Akt1 and p40phox. Data are expressed as the relative percentage to the following groups: (A) nontargeting control siRNA (CTR), (B–D) IgG-K. pneumoniae-stimulated cells pretreated with siRNA CTR, or (E) IgG-K. pneumoniae-stimulated cells. Data represent the mean ± se from at least 3 independent experiments (A–D) or a representative blot from 2 experiments (E); in the latter, relative densitometric ratios are noted below the Akt1 band. ^#P < 0.05 versus unstimulated cells pretreated with siRNA CTR; *P < 0.05 versus unstimulated (A) or IgG-K. pneumoniae-stimulated (B and C) cells pretreated with siRNA CTR. p, phospho; IP, immunoprecipitation; WB, Western blot.