The compiled results of the in vitro coupled transcription/translation analysis.
A. Denaturing gel analysis of [35S]methionine labelled translation products visualized by autoradiography. Samples A-G correspond to the in vitro-translated products obtained using Quick CoupledTranscription/Translation System (Promega) as described in "Methods" with plasmid DNA templates containing human PPARδ with the 5'-UTRs denoted A-G in Figure 1B. The composition of untranslated exons in the 5'-UTRs are: (A) Exon 1, (B) Exons 1+2, (C) Exons 1+2+3, (D) Exons 1+2+2e+3, (E) Exons 1+2+spliced 2a(90 bp)+3, (F) Exons 1+2+2a, (G) Exons 1+2+2a+3. BL is a negative control sample without template and Luc is a positive luciferase control sample.
B. Western blot analysis of the products of the translation reactions probed with the polyclonal PPARδ specific antibody IMG-3297. The sizes of standard molecule markers are given on the left side (A and B).
C. Quantification of the [35S]methionine labelled band intensities was performed on the autoradiographic (grey bars) and Western blot (white bars) bands using the NIH ImageJ software. The relative intensities of signals (% Area) were calculated setting the signal of construct A to 100%. The number of upstream AUGs and the total length of each 5'-UTR is given below each bar of calculated signal.