Trans-activation activity of Pip is affected by mutations in putative α-helices 300–314 and 325–335. NIH 3T3 cells were cotransfected with 300 ng of a κE3′-driven luciferase reporter alone (open columns) or in the presence of 50 ng of PU.1 (filled columns), together with 50 ng of wt or Pip mutants expression vectors, respectively (A and B). Synergistic transcriptional activation observed when both PU.1 and wt Pip were cotransfected was taken as 100%. (C) The ability of PU.1 and wt Pip (50 ng each) to induce luciferase activity was measured in the presence of 50 ng of cotransfected mutant Pip expression vector. Relative luciferase activity obtained in the absence of mutant Pip (column 1) was used as control. (A) Column 1, wt; column 2, Δ374–450; column 3, Δ409–450; column 4: ΔPip. (B and C) Column 1: wt; column 2: K94E; column 3, E305K; column 4, E305N K306N; column 5, E305K K306D; column 6, E312N R313E; column 7, G314V; column 8, K327G; column 9, R328K; column 10, R328E; column 11, R328Q.