Both N17Rac and N17Cdc42 block

C albicans–induced MAPK/ERK activation and PMN activity against C albicans. (A) PMNs were infected with recombinant vaccinia virus expressing dominant-negative Rac (N17Rac), Cdc42 (N17Cdc42), Rho (N19Rho), or an irrelevant protein (CD56) for 4 hours at 37°C. (A) The infected PMNs were treated withC albicans, and samples were collected at 0 minutes (lanes 1, 3, 5, 7, 9) and at 5 minutes (lanes 2, 4, 6, 8, 10). Cell lysates were probed with antiactive MAPK (top panel) and reprobed with the marker protein anti-CD56 to demonstrate successful viral infection (middle panel). The blot was again stripped and reprobed with anti-panMAPK/ERK for equal loading (lower panel). (B) Aliquots from the same PMNs were also tested for their ability to inhibit C albicans growth. Results shown are representative of 3 separate experiments. (C) PMNs were infected with CD56, N17Rac, or N17Cdc42 for 2 to 6 hours at 37°C. The infected PMNs were then collected at each time point, and cell lysates were probed with anti-Rac1 (top panel), CD56 (middle panel), or Cdc42 (bottom panel) as indicated. The myc-tagged virally expressed proteins were differentiated from endogenous Rac1 and Cdc42 by the appearance of a slower migrating band (indicated by open arrow). (D) PMNs were infected with CD56, N17Rac, or N17Cdc42 for 15 minutes at 37°C and then reinfected with either constitutively active V12Rac1 or V12Cdc42 for 4 hours at 37°C. The infected PMNs were then treated with C albicans,and samples were collected at 0 minutes and at 5 minutes, and cell lysates were probed with antiactive MAPK (top panel) or CD56 for expression of the marker protein (middle panel) or anti-panERK for equal loading (bottom panel).