Vaccinia viral expression of dominant-negative MEK 1 but not Ras blocks

C albicans–induced ERK activation. (A) PMNs were infected with recombinant vaccinia virus expressing dominant-negative MEK1, dominant-negative Ras (N17Ras), or an irrelevant protein (CD56) for 4 hours at 37°C. The infected PMNs were then incubated withC albicans, and samples were collected at 0 minutes (lanes 1, 3, 5, 7) and 5 minutes (lanes 2, 4, 6, 8). Cell lysates were then immunoblotted with antiactive MAPK (upper panel). The blot was then stripped and reprobed with the marker protein anti-CD56 to demonstrate that the viral infection was successful (middle panel). The blot was also reprobed with anti-panERK for equal loading (lower panel). (B) Aliquots of PMNs from the same experiment as in panel A were tested for growth inhibition of C albicans. Results shown are representative of 3 separate experiments. (C) PMNs either mock infected or CD56 infected at MOI of 1:6 for 12 hours or 24 hours were collected and analyzed for apoptosis by annexin V–PE binding. The percentage of apoptotic cells is indicated.