Figure 7.
Filamin B serves as a scaffold for JNK cascade members. (A) M2 cells complemented with HisMax-c-filamin B were treated with IFNα. Cell lysates were subjected to pull-down with NTA resins followed by immunoblot analysis. The same precipitates and lysates were also probed with anti-p-JNK or anti-JNK antibody. (B) M2 cells transfected with an empty or HisMax-c-filamin B vector were cultured for 1 h with IFNα. Cell lysates were subjected to gel filtration on a Sephacryl S-300 column (0.7 × 28 cm) equilibrated with 20 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 7% glycerol. Fractions of 0.2 ml were collected and subjected to immunoblot with anti-Xpress antibody or antibody against Rac1, MEKK1, MKK4, or JNK. The size makers used were a, thyroglobulin (Mr = 669,000); b, β-amylase (200,000); and c, albumin (66,000). (C) FLAG-Rac1, HA-MEKK1, Myc-MKK4, or HA-JNK1 was expressed in HeLa cells with or without HisMax-c-filamin B. Cells were fixed and stained with anti-FLAG, anti-Xpress, anti-HA, or anti-Myc antibody. Bars, 10 μm. Similar data were obtained by at least three independent trials of each experiment set.