Rb enhances C/EBPϵ transactivation. (A) Soas-2 cells were cotransfected with a G-CSFR promoter reporter vector (pTK81G/G-CSFR, 1 μg) and one or more of 4 constructs (1 μg each) that express C/EBPϵ, wild-type Rb (Rbwt), a constitutively active mutant Rb (Rb5C), or an inactive mutant Rb (Rb16). (B) Soas-2 cells were cotransfected with a mim-1 promoter reporter vector (pGL3B/mim-1, 1 μg) and the various expression constructs as described for panel A. All transfections included the pRL-SV40 vector (0.2 μg) that served as internal control for transfection efficiency. Results represent the mean of triplicate transfections. The experiments were repeated 3 times. (C) NIH 3T3 cells stably transfected with either an inducible C/EBPϵ gene (pMTϵ) or C/EBPα gene (pMTα) were incubated either without (–) or with (+) zinc (16 hours) and analyzed by Western blot using antibodies to C/EBPϵ and C/EBPα. The blots were stripped and rehybridized with a GAPDH antibody. (D) The pMTϵ and pMTα cells were transiently transfected with either an empty vector (ev) or a Rb expression vector (Rb) and treated with zinc for 16 hours. cDNA from these cells was subjected to real-time RT-PCR with specific primers and probes for B9 and 18S. The results are expressed in arbitrary units as a ratio of either B9 transcripts/18S transcripts (each value represents the mean of 3 measurements of the sample).