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. 2008 Jun 6;283(23):15903–15911. doi: 10.1074/jbc.M710006200

FIGURE 5.

FIGURE 5.

JNK3 recruits MKK4 to β-arrestin-2. A, constructs expressing HA-ASK1 and FLAG-β-arrestin-2 were introduced into COS-7 cells. In the control an empty vector was transfected in place of the β-arrestin-2 expression vector. β-Arrestin-2 was immunoprecipitated with the M2 antibody (M2-IP) and ASK1 present in the precipitates detected with anti-HA antibody. The expression of ASK1 and β-arrestin-2 was examined by immunoblotting the lysates with anti-HA and M2 antibodies, respectively. B and C, constructs expressing GST or GST-MKK4 were introduced into COS-7 cells together with an expression vector for HA-ASK1 (B) or HA-JNK3 (C). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the ASK1 or JNK3 present in the pull-down (PD) was examined by immunoblot using anti-HA antibody. The expression of the MKK4, ASK1, and JNK3 was examined by immunoblotting the lysates with anti-GST and anti-HA antibodies, respectively. D, constructs expressing GST-tagged MKK4 were introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 and either HA-JNK3 or HA-ASK1. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-down (PD) was examined by immunoblot using M2 antibody. The expression of the MKK4, β-arrestin-2, ASK1, and JNK3 was examined by immunoblotting the lysates with anti-GST, M2, and anti-HA antibodies, respectively. E, construct expressing GST-MKK4 was introduced into COS-7 cells together with an expression vector for FLAG-β-arrestin-2 and the indicated JNK mutants. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the β-arrestin-2 present in the pull-down (PD) was examined by immunoblot using M2 antibody. The expression of the MKK4, β-arrestin-2, and the JNK mutants was examined by immunoblotting the lysates with anti-GST, M2, and anti-HA antibodies, respectively. * indicates nonspecific bands detected by the HA antibody. Experiments were performed either two or three times and representative immunoblots are shown.