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VE-cadherin protein levels in monolayer HUVEC cultures

Analysis of c-met and VE-cadherin mRNA in HUVECs grown in different culture conditions

We used a semiquantitative analysis by RT-PCR to determine whether the culture condition–dependent differences in the c-met and VE-cadherin protein levels are accompanied by differences in the corresponding mRNA levels. We used serial dilutions of the reverse transcriptase reaction as a template for PCR . In our experimental conditions the quantity of amplified product decreased in relation to template concentration. This indicated that the PCR yield reflected the changes in the abundance of the corresponding mRNA. Therefore, these conditions were used for semiquantitative evaluation of the relative levels of 3 mRNAs: c-met, VE-cadherin, and β-actin.

Figure 4.

Figure 4.

RT-PCR analysis of c-met, VE-cadherin, and β-actin mRNA in HUVECs grown in different culture conditions. Total RNA extracted from confluent and sparse HUVECs and from the HUVECs grown in 3-dimensional collagen gel was reverse transcribed with AMV reverse transcriptase and amplified using PCR, as described in “Materials and methods.” Amplified products were analyzed on a 1.5% agarose gel after staining with ethidium bromide. Figures on the top indicate the dilution of the single-stranded cDNA template used for the PCR. Results are representative of 4 independent experiments.