FIG. 4.

Generation of transgenic mice that specifically express either WT or phosphorylation-resistant RhoA in arterial smooth muscle. (A) Transgene constructs for WT RhoA (RhoA^wt) and phosphorylation-resistant RhoA (RhoA^A188). Arrowheads, primer set used for genotyping and RT-PCR analysis in panel C; gray bar, SpeI-SpeI fragment recognized by the XbaI-HindIII probe (open bar) in the Southern blotting analysis in panel B. bGH, bovine growth hormone polyadenylation signal; 4F2, human 4F2 enhancer. (B) Genomic Southern blot analysis was performed with the F1 and F2 offspring of the RhoA^WT (line 39) and RhoA^A188 (line 30) transgenic mice. The transgenic vector (A) was used to quantify the copy number (first and second lanes). (C) Transgene mRNA expression was assessed by semiquantitative RT-PCR analysis of aortas between different founder lines (F2 offspring) of RhoA^WT-Tg and RhoA^A188-Tg mice. (D) Representative immunostaining with anti-myc-tag antibody showing transgene protein expression in NTg, RhoA^WT-Tg (line 39), and RhoA^A188-Tg (line 30) mouse heart and aorta sections (brownish signal). L, lumen. Bars, 50 μm. (E) Immunoblot with anti-RhoA and anti-myc-tag antibodies showing the ∼24-kDa transgene product expression in NTg, RhoA^WT-Tg, and RhoA^A188-Tg mouse aortas. Arrowhead (myc-RhoA), transgene product; arrow, endogenous RhoA. (F) Immunoblot showing the P-moesin level in NTg, RhoA^WT-Tg, and RhoA^A188-Tg mouse aorta lysates. Values are means ± SEM for triplicates. *, P < 0.05.