Helicase-Like Transcription Factor (Hltf) Regulates G2/M Transition, Wt1/Gata4/Hif-1a Cardiac Transcription Networks, and Collagen Biogenesis
Figure 2
Western blot, ChIP-PCR and competitive RT-PCR.
Panel A, Western blotting confirmed the presence of full-length Hltf protein (116-kDa between molecular markers 120 and 100 kDa) in hearts of control mice (lane 1), compared with the absence of protein expression in hearts of Hltf null mice (lane 2). Panel B, electrophoretic resolution of a single population of amplicons from touchdown PCR of ChIP confirmed Hltf bound the transcriptionally active regulatory regions of the promoters of Gata4 (248-bp; lane 1) and Myh7b/miR499 (228-bp; lane 2). Water blank control (lane 3) and molecular ladder (bp) are shown. Panel C, electrophoretic resolution of a single population of amplicons from touchdown PCR of ChIP confirmed Hltf bound the putative promoter of Hif-1a (600-bp, lane 1). Water blank control (lane 2) and φX174 markers are included. Panel D, analysis of competitive RT-PCR products by electrophoresis for the presence/absence of exon 7 in heart (lanes 1-2) and brain (lanes 3-4) revealed the ratio of amplicons without exon 7 (293-bp) to amplicons with exon 7 (390-bp) was the same in the presence (lane 1, 3) and absence (lane 2, 4) of Hltf. The water blank control (lane 5) and φX174 markers are added for completeness. The identity of each population of amplicons was verified by double-stranded sequencing.