FIG. 5.

(A) Overexpression of ERK inhibits TEL's trans-repressional ability. NIH 3T3 cells were transfected with 1 μg of the pGL2-754TR reporter plasmid alone or along with 0.5 μg of pME18S-FLAG-TEL or pME18S-FLAG-S22/S238 with or without 0.5 μg of ERK expression plasmid and cultured in DMEM containing 10% FCS for 48 h. (B) Activation of endogenous ERK also inhibits TEL's trans-repressional ability. NIH 3T3 cells were transfected with 1 μg of the pGL2-754TR reporter plasmid alone or along with 1 μg of pME18S-FLAG-TEL or pME18S-FLAG-S22/S238. After 48 h, the cells were incubated in DMEM containing 10% FCS with or without recombinant human EGF for 2 h before harvest. (C) Simultaneous replacement of Ser²¹³ and Ser²⁵⁷ residues with glutamates eliminates TEL's trans-repressional ability. NIH 3T3 cells were transfected with 1 μg of the pGL2-754TR reporter plasmid alone or along with wild-type TEL or various kinds of TEL glutamate mutant expression plasmids and incubated for 48 h before harvest. (D) The glutamate mutant E213/257 shows a dominant-negative effect on TEL-mediated transcriptionalrepression. NIH 3T3 cells were transfected with 1 μg of the pGL2-754TR reporter plasmid alone (lane 1) or along with 0.1 μg of pME18S-FLAG-TEL (lanes 2 to 8). In lanes 3 to 8, 0.05, 0.5, and 0.9 μg of pME18S-FLAG-E213/257 or pME18S-EVI-1 were cotransfected as well. NIH 3T3 cells were also transfected with 1 μg of the pGL2-754TR reporter plasmid along with 0.05, 0.5, and 0.9 μg of pME18S-EVI-1 alone (lanes 9 to 11). Bars show luciferase activities relative to the level observed when control plasmid pME18S was cotransfected, and average results of duplicate experiments are presented.