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Fig. 10. Western blot analysis of Akt phosphorylation in cells transfected with siRNAs, and HUVEC migration rescue experiment with myr-Akt. (A) HUVECs in six-well plates were transfected with NC, si1 and si3 on day 0, split on day 1, serum-starved with EBM-2 supplemented with 0.5% serum on day 2 and stimulated with EBM-2 supplemented with VEGF (5 ng/ml) and 0.5% serum or with EGM-2 MV for 30 minutes on day 3. (B) HUVECs in 10-cm plates were transfected with NC, si1 and si3 on day 0, split onto 2 plates in the early morning of day 1 and infected with myr-Akt adenovirus in the late afternoon. EGM-2 MV was changed 6 hours later, EBM-2 supplemented with 2.5% serum was changed in the evening of day 2 and migration assay was done on day 3 by stimulating with DMEM supplemented with VEGF (10 ng/ml) and 0.5% serum. Data from three experiments are shown as the mean ± s.d. All indicated P values for the comparisons were obtained using two tailed Student's t-test.
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