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Fig. 3. Effect of wild-type and {Delta}bas TAL-1 overexpression on in vitro angiogenesis. (A) Western blot analysis showing ectopic overexpression of wild-type (wt) and {Delta}bas TAL-1 protein in adenovirus-infected HUVECs. ECs were co-infected with Ad-Tet-Off and one of the following: Ad-TRE-wtTAL-1, Ad-TRE-{Delta}bas or the control Ad-TRE-LacZ. Whole cell lysates were prepared 20 hours after infection. Note the intensities of wt and {Delta}bas TAL-1 detected in 1 µg of whole cell lysate derived from infected cells as compared to that of endogenous TAL-1 detected in 30 µg of whole cell lysate derived from Ad-LacZ-infected cells (LacZ) or non-treated cells (NT). (B) Electrophoretic mobility shift analysis (EMSA) of TAL-1 DNA binding activity. A double stranded 32P-labeled oligonucleotide probe containing the TAL-1 E-box consensus sequence was incubated with nuclear extracts from HUVECs. (Left) EMSA showing the binding of TAL-1/E endogenous heterodimers to the TAL-1 E-box in nuclear extract from HUVEC (10 µg). The specificity of the interaction was controlled by adding a 100-fold excess of unlabeled TAL-1 E-box oligonucleotides (comp). Additionally, the presence of TAL-1/E heterodimers was verified by supershifting the complexes with an anti-TAL-1 antibody ({alpha}TAL) or disrupting them with an anti-E47 antibody ({alpha}E47). An irrelevant monoclonal antibody (12CA5) was added as a negative control. (Right) Ectopic TAL-1 DNA binding on E-box sequences was monitored in the presence or absence of a supershifting anti-TAL-1 mAb. Experimental conditions precluded the detection of endogenous TAL-1/E heterodimers in control LacZ-infected HUVECs (only 3 µg of nuclear extract was used). Note the absence of TAL-1/E DNA complex in nuclear extract from ECs infected with Ad-TRE-{Delta}bas. (C) The effect of wt or {Delta}bas TAL-1 overexpression on in vitro angiogenesis. Angiogenesis was monitored using adenovirus-infected ECs plated on Matrigel. Representative photographs of HUVECs after 90 minutes and 20 hours of plating are shown.





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