Fig. 5. Deletion of Cx37 or Cx40 does not alter mRNA levels of non-ablated connexins in aortic endothelium. (A) RT-PCR for Cx37, Cx40, Cx43 and GAPDH mRNA was performed with RNA isolated from aortic endothelium. Amplicons were of the predicted size and depended on reverse transcription (RT). Cx37 and Cx40 amplicons were absent in samples derived from Cx37^–/– or Cx40^–/– mice, respectively. (B) Semiquantitative RT-PCR was performed. Connexin signals were normalized to GAPDH signals. (C) Quantification of Cx37 signals obtained from groups of wild-type and Cx40^–/– animals yielded similar amplification curves. (D) Mean Cx37, Cx40 and Cx43 mRNA levels (relative to wild-type levels) are plotted. Wild-type signals for each connexin mRNA were assigned the value of 1.0. Error bars represent s.e.m. There were no significant changes in the levels of non-ablated connexin mRNAs present in Cx37^–/– and Cx40^–/– aortic endothelium (P>0.05). (E) Endothelial or whole aorta RNA preparations (equivalent lengths of aorta) from wild-type mice were analyzed by RT-PCR for smooth-muscle actin mRNA to test for contamination of endothelial RNA fractions with medial layer RNA. A 1/500 dilution of whole aorta cDNA yielded a similar amplification curve to that of undiluted endothelial cDNA. Contamination was therefore approximately only 0.2%. Cx40 RNA levels were the same in each preparation, whereas Cx37 RNA levels were slightly higher in the whole aorta sample.