Figure 2.
Macrophage Rac2 (Ras-related C3 botulinum toxin substrate 2) regulates NOX2 (NADPH oxidase 2)-derived reactive oxygen species (ROS). (A and B) Membrane-derived ROS generation in THP-1 cells treated with the indicated concentration of cigarette smoke extract (CSE) for 3 hours (n = 3) (A) and THP-1 cells expressing scrambled or gp91phox siRNA treated with vehicle or CSE (5%) for 3 hours (n = 3) (B). (B, inset) Immunoblot analysis confirming gp91phox silencing. (C and D) Immunoblot analysis of NOX2 complex proteins in isolated membrane or cytosol fractions from MH-S cells treated with the indicated concentrations of CSE (C) and THP-1 cells expressing scrambled or Rac2 siRNA treated with vehicle or CSE (D). (E) Immunoblot analysis of THP-1 cells expressing scrambled or p67phox siRNA treated with vehicle or CSE. (F) Histidine (His) pull-down in MH-S cells expressing empty or p67phox-V5-His and treated with vehicle or CSE and (G) quantification. (H) Immunoblot analysis of BAL cells from air- or CS-exposed mice (n = 3). (I and J) Rac2 activity in BAL cells from air- and CS-exposed mice infected with vehicle or Streptococcus pneumoniae (n = 4) (I) and mice treated with control or clodronate liposomes and infected with vehicle or S. pneumoniae (n = 4) (J). (K) Rac2 membrane activity in BAL cells from control and anti-Ly6G–treated mice infected with vehicle or S. pneumoniae (n = 5–6). **P < 0.001; ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for A, B, and I–K. Results from A, B, and J were repeated three times; immunoblot analyses were performed three times, with representative immunoblots shown in C–F and H; I was conducted at least five times. Cyto = cytosol; IB = immunoblot; Mem = membrane; PD = pull-down; Scr = scrambled; S.p. = Streptococcus pneumoniae.