Fig. 2.

Wnt3A increases p-Tyr42 RhoA and both p-Ser9 and p-Tyr216 GSK-3β levels. (A) HEK293T cells (2–5x10⁵) were treated with H₂O₂ (100 μM) for the indicated periods and p-Tyr42 RhoA was detected by western blotting. (B) HEK293T cells were transfected with si-RhoA (10 nM) for 72 h and then transfected with RhoA WT, Y42E and Y42F plasmid DNA (4 μg) for 18 h. Produced superoxide was detected with hydroethidine as above. (C) Cells were pretreated for 1 h with NAC (10 mM), DPI (10 μM), and apocynin (10 μM) and the indicated proteins were detected by western blotting. (D) Cells were pretreated with PP2 (10 μM) for 1 h and then treated with Wnt3A (30 ng/ml) for 2 h and subsequently, the indicated proteins were detected by western blotting. (E) Real time activation of Src in membrane and cytosol of 293 A cells was measured using FRET methods. (F) HEK293T cells were pretreated with PF431396 (10 μM), an inhibitor of PYK2, stimulated by Wnt3A and then p-Tyr216 and p-Ser9 GSK-3β were determined. (G) Cells were transfected with si-Src and stimulated by Wnt3A. p-Tyr216 GSK-3β was determined by immunoblotting. (H) HEK293T cells were pretreated with Y27632 (10 μM) for 1 h and then treated with Wnt3A (30 ng/ml) for 2 h; p-Tyr216 GSK-3β level changes were detected by western blotting. (I) 293 A cells were incubated for 12 h without serum and were then treated with Wnt3A (50 ng/ml) for 30 min. Cells were then fixed using 4% PFA and p-Ser9 GSK-3β (green: Alexa-488) and p-Tyr216 GSK-3β (red: Alexa546) levels were visualized with the respective secondary antibodies. (J) Cells were stimulated with Wnt3A (30 ng/ml) for 2 h, RhoA and p-Tyr42 RhoA were immunoprecipitated and co-immunoprecipitated ROCK1 and ROCK2 were detected with western blotting. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)