MCP-1 synthesis in cells expressing dominant negative MKK6(Ala), MKK6 wild-type, or constitutively active MKK6(Glu) in the presence or absence of TNF-. Cells were transfected in a 1:3 ratio with pGreenLantern expressing GFPS65T and plasmids expressing either empty expression vector KRSPA, KRSPA MKK6(Ala), KRSPA MKK6 wild-type, or KRSPA MKK6(Glu). (A) Cells transfected with empty expression vector or dominant negative MKK6, ie, MKK6(Ala), were either left unstimulated or stimulated with 2 ng/mL TNF- for 12 hours. Successfully transfected cells were analyzed for MCP-1 synthesis by flow cytometry as described in Materials and Methods. Flow cytometry profiles of one representative experiment are shown. Open profiles represent isotype controls, shaded profiles represent cells labeled for MCP-1 expression. Bold letters indicate the mean fluorescence intensities; additionally, percentages of MCP-1–positive cells are given. (B) HUVEC transfected with empty expression vector, wild-type MKK6, or a constitutively active mutant of MKK6, ie, MKK6(Glu), were exposed to a limited concentration of TNF- (0.2 ng/mL) and evaluated for MCP-1 expression as indicated.