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Fig. 1. TAL-1 protein expression in human endothelial cells. (A) pp48-50 is the only TAL-1 protein species detected in human proliferating endothelial cells (ECs). (Left) TAL-1 expression was assessed in whole cell lysates (30 µg) by immunoblotting with the 3BTL73 monoclonal antibody directed against the C terminus of the human TAL-1 protein (top). The blot was reprobed with an anti-GAPDH rabbit antiserum to evaluate protein loading. Expression was assessed in the following cells: K562, a human erythroleukemic cell line; ECV 304, an epithelial cell line established from a human bladder carcinoma; HUVEC, ECs derived from umbilical cord; HDMEC, ECs derived from dermal microvessels; HMEC-1, a cell line derived from HDMECs immortalized with large T antigen (Ades et al., 1992) and BMEC-1, a cell line established from bone marrow ECs immortalized with large T antigen (Candal et al., 1996). (Right) Expression of TAL-1 protein was assessed in circulating endothelial cells derived from umbilical cord blood (UCB-EC) at day 25 of culture (see Materials and Methods). The blot was reprobed for FLI-1 and VE-cadherin protein expression, two endothelial-specific markers, and for GAPDH to monitor loading. (B) Down-regulation of tal-1 expression in quiescent non-activated HUVECs. Proliferating HUVECs (Expo) were grown to confluence and were maintained either in complete medium for an additional 8 hours (Conf) or in medium containing only 5% FCS for 3 hours and 8 hours. (Left) Whole cell lysates were prepared at the indicated times after starvation and TAL-1 expression was monitored by western blot analysis using the 3BTL73 anti-TAL-1 monoclonal antibody. The same blot was reprobed with an anti-GAPDH rabbit antiserum for normalization of protein levels. TAL-1 and GAPDH proteins were both quantified by densitometry and TAL-1 protein content relative to GAPDH in one of two representative experiments is shown. The TAL-1 content from exponentially growing HUVECs (Expo) was arbitrarily set at 1. (Right) tal-1 mRNA expression was monitored by real-time PCR. The tal-1 mRNA signal was normalized to that of GAPDH. Data shown are the means ±s.d. of three independent RT-PCR experiments. tal-1 mRNA content from exponentially growing HUVECs was arbitrarily set at 1.
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