Effect of dominant-negative Rac or RhoGDI expression on agonist-elicited superoxide production by COS-phoxcells.
COS-phox cells were transiently transfected with expression vectors encoding dominant-negative Rac1(T17N), Rac2(T17N), or RhoGDI. Control cells were transfected with empty pRK5 vector. Cells were analyzed 21 hours after transfection. (A) Whole-cell lysates were separated by 12% SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal Ab to Rac1 or polyclonal Ab to RhoGDI. Transgenic Rac1N17 migrated more slowly than endogenous Rac1 owing to an N-terminal Myc-epitope tag, and it migrated as a doublet owing to the presence of both isoprenylated and nonisoprenylated forms. Transgenic RhoGDI migrated more slowly than endogenous RhoGDI owing to 5 extra N terminal amino acids. Blots are representative of 4 or more independent assays. (B) Cells were stimulated with 0.4 μg/mL PMA and assayed for superoxide production by the ferricytochrome c reduction assay. The activity of the control, empty vector–transfected cells was 12.2 ± 2.8 nmol O2− per 107cells per minute. Bars represent mean ± SD; n ≥ 4. *P < .01 by paired t test.
