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

FIGURE 6.

FIGURE 6.

MKK4 is recruited to the β-arrestin-2 scaffold complex via its N-terminal D-domain. A, constructs expressing GST, GST-MKK4, or GST-MKK4 (L44A/L46A) were introduced into COS-7 cells together with an expression vector for HA-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and JNK3 present in the pull-down (PD) was examined by immunoblot using anti-HA antibody. The expression of the MKK4 and JNK3 was examined by immunoblotting the lysates with anti-GST and anti-HA antibodies, respectively. B, constructs expressing GST-MKK4 or GST-MKK4 (L44A/L46A) were introduced into COS-7 cells together with expression vectors for HA-JNK3 and FLAG-β-arrestin-2. 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, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST, anti-HA, and M2 antibodies, respectively. C and D, constructs expressing GST, GST-JNK3, or GST-JNK3(CDmut) were introduced into COS-7 cells together with expression vectors for either FLAG-β-arrestin-2 (C) or Myc-tagged MKK4 (D). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and β-arrestin-2 and MKK4 present in the pull-down (PD) were examined by immunoblot using M2 and anti-Myc antibodies, respectively. The expression of the JNK3, β-arrestin-2, and MKK4 was examined by immunoblotting the lysates with anti-GST, M2, and anti-Myc antibodies, respectively. E, constructs expressing GST-tagged MKK4 were introduced into COS-7 cells together with expression vectors for FLAG-β-arrestin-2 and either HA-JNK3 or HA-JNK3 (CDmut). 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, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-GST, anti-HA, and M2 antibodies, respectively. * indicates nonspecific bands detected by the HA antibody. Experiments were performed either two or three times, and representative immunoblots are shown.