Figure 2

Erk2 and p38α, but not Jnk1 interacted with ArhGAP9 WW domain. a. Specificity of Erk2 and p38α binding to the WW domain of ArhGAP9. MAP kinases, Erk2-Flag, p38α-Flag or Jnk1-Flag, were transiently expressed in 293T cells and the lysates used for in vitro binding assay with (i) ArhGAP9-WW-GST or (ii) GST control, followed by western blotting with α-Flag. (iii) Total cell lysates were separated by SDS-PAGE and immunoblotted with α-Flag. b. Full length Erk2 and p38α interact with ArhGAP9 in vitro. Flag-tagged ArhGAP9 was expressed in 293T cells and the lysates were incubated with immobilized (i) Erk2-GST, (ii) p38α-GST or (iii) GST alone as a control. Bound Erk2 and p38α was detected by western blotting with α-Flag. (iv) Total lysates were immunoblotted with α-Flag. c. Interaction of ArhGAP9 with MAP kinases in vivo. ArhGAP9 and MAP kinases, Erk2-Flag or p38α-Flag were expressed in 293T cells. The lysates were immunoprecipitated with α-Flag, followed by western blotting with (i) α-ArhGAP9 or (ii) α-Flag. (iii, iv) Immunoblotting of total cell lysates with α-ArhGAP9 or α-Flag. d. Erk2 and p38α interact specifically with the WW domain of ArhGAP9 but not ArhGAP12 or Nedd4. MAP kinases Erk2-Flag, p38α-Flag or Jnk1-Flag was expressed in 293T cells and the lysates were incubated with GST fusion proteins of the WW domains of ArhGAP9, ArhGAP12 or Nedd4, or GST alone immobilized on glutathione Sepharose beads, as indicated in the figure. (i) Specifically bound proteins were detected by western blotting with α-Flag. (ii) Total cell lysates were immunoblotted with α-Flag. e. Erk2 and p38α do not interact with mouse ArhGAP9 in vitro. Flag-tagged Erk2, p38α or Jnk1 plasmid was expressed in 293T cells. Immobilized GST proteins for (i) WW domain of human ArhGAP9, (ii) N-terminal fragment of mouse ArhGAP9 (residues 1–350) or (iii) GST alone was incubated with the lysates. Bound Erk2, p38α or Jnk1 was detected by western blotting with α-Flag. (iv) Total cell lysates were immunoblotted with α-Flag. f. Erk2 interact with human but not mouse. Human or mouse ArhGAP9-Flag was expressed in 293T cells, individually or together with Erk2-HA. The lysates were immunoprecipitated with α-Flag. (i) Bound Erk2 was detected by western blotting with α-HA. (ii) Immunoprecipitated human or mouse ArhGAP9 was detected by α-Flag. Total cell lysates were immunoblotted with (iii) α-Flag or (iv) α-HA.