Figure 8.

Filamin B promotes sequential activation of JNK cascade members. (A) Myc-MKK4 was expressed in HeLa cells with HisMax-c-filamin B, HA-MEKK1, or both. Cells lysates were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot with respective antibodies. (B) JNK1-Myc-His was expressed in HeLa cells with HA-MEKK1, HisMax-c-filamin B, or both. Cell lysates were assayed for the phosphorylation of cJun, followed by immunoblot with anti-p-cJun antibody. (C) JNK1-Myc-His was expressed in HeLa cells with Myc-MKK4, HisMax-c-filamin B, or both. (D and E) HisMax-tagged full-length filamin B or its mutant form having in-frame deletion of R20 (ΔR20), R21 (ΔR21), or both R20 and R21 (ΔR20-21) was expressed in HeLa cells with Myc-JNK1 (D) or Myc-MEKK1 (E). Cell lysates were then subjected to immunoprecipitation with anti-Myc antibody. (F) HisMax-tagged filamin B, ΔR21, or ΔR20-21 was complemented to M2 cells. After incubation with IFNα for 1 h, cell lysates were subjected to immunoblot with anti-p-JNK, anti-JNK, or anti-Xpress antibody. (G) HeLa cells were transfected with an empty (control) or HisMax-R20-24 vector. After incubating them with IFNα for various periods, cell lysates were subjected to immunoblot with anti-p-JNK, anti-JNK, anti-filamin B, or anti-Xpress antibody. In A–C, the numerals indicate the quantified band intensities relative to that seen with MKK4 or JNK alone. In F, the numerals indicate the quantified band intensities relative to that seen with transfection of an empty vector. All the numerals are representative of three independent experiments.