Fig. 2. Time course of the sodium orthovanadate (Na³VO⁴)–induced tyrosine phosphorylation of a set of substrates (  A ), phospholipase Cγ-1 (PLCγ-1) (  B ), and p44/p42 mitogen-activated protein kinase (MAPK) (  C ) in rat aortic smooth muscle. Rat aortas were homogenized in lysis buffer before or 1, 2, 5, 10, 20, 30 min after application of Na³VO⁴(10⁻⁴m). Some aortas were incubated with genistein (5 × 10⁻⁵m) for 15 min, and were then homogenized 20 min after being exposed to Na³VO⁴. Protein tyrosine phosphorylation was detected using Western blotting with specific tyrosine phospho-antibodies. The time courses of the Na³VO⁴-induced tyrosine phosphorylation (  i.e. , the band densities) of all the substrates (especially proteins with molecular weights of 42, 71, 88, 116, and 155 kd), including PLCγ-1 and p42MAPK, was similar to that of the Na³VO⁴induced-contraction, approximately reaching peak level 10 min after application of Na³VO⁴, but no significant change in the density of tyrosine phospho-p44MAPK band was observed with Na³VO⁴treatment (  D ). The Na³VO⁴-induced tyrosine phosphorylation of all the proteins was significantly reduced in the presence of genistein. The image was representative of four independent experiments.