Figure 9.

Filamin B is required for IFNα-induced activation of JNK cascade. (A) HeLa cells transfected with shRNAs or shControl were incubated for 1 h with or without IFNα. Cell lysates were immunoprecipitated with anti-MEKK1 antibody. Precipitates were then subjected to in vitro kinase assay for MEKK1 by using GST-MKK4 as a substrate. (B) Cells were incubated as in A, and their lysates were immunoblotted with anti-p-MKK4. (C) A vector expressing Rac1/G12V was transfected to HeLa cells that had been transfected with shRNAs or shControl. Cell lysates were assayed for cJun phosphorylation. (D–F) HeLa cells transfected with shRNA specific for Rac1 (D), MEKK1 (E), or MKK4 (F) were incubated for 1 h with or without IFNα. Cell lysates were then subjected to immunoblot with anti-p-JNK or anti-JNK antibody. In A–F, the numerals indicate the quantified band intensities relative to that seen with shControl only and are representative of three independent experiments. (G) HeLa cells transfected with shRNA specific for MKK6 or Cdc42 were incubated for 1 h with or without IFNα. Cell lysates were then subjected to immunoblot with respective antibodies.