Analysis of CCR8 mRNA expression in HUVECs.
(A) PCR amplification was performed on RNAs or cDNAs of HUVECs grown in complete medium. Shown is a representative experiment of 4 performed. RT-PCR was performed with CCR8 primers on RNA samples (absence of reverse transcription) (control), and cDNA samples (CCR8). Also shown is a representative reaction with GAPDH primers on the cDNA template (GAPDH). (B) The upper panel shows an RNase protection assay performed using 200 ng Th2 positive control total RNA (lane 1), 2 μg yeast total RNA (lane 2), and 10 μg of total RNA extracted from HUVECs (lane 3) using the hCR5 multiprobe from Pharmingen. Approximately 500 cpm of 32P-labeled multiprobe were loaded on lane 4, and the corresponding RNA species marked on the right-hand side. The arrow shows the CCR8 message visible in lane 1 and lane 3. The RNA species in lane 4 (probe) show, as expected, a slightly higher molecular weight than in lane 1 and 3, due to the presence of flanking regions in the in vitro transcript. The bottom panel shows the L32 and GAPDH RNA species. The dried gel was subjected to autoradiography at −80°C and developed with an automatic Kodak developer. The upper panel was exposed for 16 hours and the lower panel for 20 minutes.
