FIG. 5.

(A) Effect of B1R on JIP1 interaction with TAK1/TAB1. Cos1 cells were transfected with pHA-TAK1 and pFlag-TAB1, different amounts of pSG-HA-B1R or pSG-HA-B1R(K149Q), and pGST-JIP1 as indicated. The correct expression of the proteins in the whole-cell extract is shown in the top gel. These extracts were used for a pull down with glutathione-Sepharose (arrow), and the proteins detected in a Western blot are shown in the middle panel. (B) Effect of B1R on the JIP1 interaction with MKK7. Cos1 cells were transfected with pFlag-MKK7, different amounts of pSG-HA-B1R or pSG-HA-B1R(K149Q), and pGST-JIP1 as indicated. The proteins in the whole-cell extract were used for a pull down with glutathione-Sepharose and Western blot analysis (middle blot). The interaction was confirmed by an alternative method using immunoprecipitation (lower right panel). For the detection of the interaction of B1R and MKK7 through JIP1, Cos1 cells were transfected with the plasmids pSG-HA-B1R and pFlag-MKK7 (1 μg) with and without pGST-JIP1. The extract was immunoprecipitated with anti-Flag (brings down MKK7 and its associated proteins), and the immunoprecipitated proteins were analyzed with an anti-HA antibody that detects B1R and anti-Flag to detect MKK7. (C) Effect of B1R on the JIP1 interaction with JNK. Cos1 cells were transfected with plasmids pFlag-JNK, different amounts of pSG-HA-B1R or pSG-HA-B1R(K149Q) and pGST-JIP1 as indicated. The expression of the proteins in the whole-cell extract is shown in the top gel, and the extract was used for a pull-down with glutathione-Sepharose and the proteins were detected in a Western blot (WB). −, without; +, with.