SRF-independent/SAP-dependent transcripts represent direct Mkl1 target genes requiring the SAP domain of Mkl1 to induce transcription from their proximal promoter. (A) The indicated promoter constructs that contained at least 500 bp upstream of the transcription start site (TSS) and were linked to the secreted alkaline phosphatase (SEAP) reporter gene, were cotransfected in HC11 cells together with an inactive Mkl1 devoid of the transactivation domain  or the FL-Mkl1 construct. SEAP activity is expressed as fold induction above the level obtained with the inactive Mkl1. In addition to Tnc, for 8 out of the 12 new promoters tested, induction greater than 2-fold (indicated by the red line) was obtained. Values are means ± SEM from three to seven independent experiments. (B) HC11 cells were cotransfected with the indicated promoter constructs that were either > 500 bp or shortened to 200 bp upstream of the TSS, and with vectors encoding the indicated mutant Mkl1 constructs. SEAP activity is normalized and expressed as in (A). Means ± SEM from at least three independent experiments and significant differences between either mutB1- and ΔSAP-Mkl1-transactivated promoter constructs or between the longer and shorter promoter constructs transactivated by mutB1-Mkl1, ***P < 0.001, **P < 0.01, *P < 0.05 are shown.