VE-cadherin expression induces Gas1 in 293 cells. The 293 cells were stimulated for 24 hours at 37°C with 1μM of the ecdysone analog muristerone A to express transfected VE-cadherin. Western blot (A) and immunofluorescence (B) analysis confirmed VE-cadherin expression and its correct localization at cell-cell contacts only in stimulated (Ecdysone +) 293 cells. Original magnification, × 400 (B). (C-D) Analysis of Gas1 up-regulation in ecdysone-treated 293 cells by Northern blot and quantitative RT-PCR. The RT-PCR data are means ± SD of 3 different experiments performed in triplicates and are expressed as relative amounts using “Ecdysone -” cell lysate as reference value. Cells transfected with the empty vector and treated with ecdysone did not show detectable VE-cadherin expression by Western blot or up-regulation of Gas1 mRNA using Northern blot analysis (not shown). For Northern blot, a comparable amount of total RNA was loaded as shown by ethidium bromide staining of the membrane. The position of 2 ribosomal RNAs (28S and 18S) is indicated.