Fig. 3. Interaction of NP60 with p38. (A) Bacterially expressed NP60, its truncated mutants, PWWP, AT-hook and NAD, ATF₂ were coated on 96-well plates and incubated with p38. The interaction was determined by ELISA. Values are means ± s.e.m. (B) Bacterially expressed GST-NP60 and His-p38 were incubated and the reaction complex was pulled down with glutathione Sepharose 4B, and detected on a blot using p38 antibody. (C) Bacterially expressed GST-NP60 and His-p38 were incubated and the reaction complex was pulled down with Ni-NTA agarose and detected by NP60 antibody on a blot. (D) Bacterially expressed GST-NP60 and its different truncated mutant proteins were incubated with His-p38 and the reaction complex pulled down with glutathione Sepharose 4B, and detected on a blot using p38 antibody. (E) Vectors that express myc-NP60 and Flag-p38 were co-transfected into 293T cells. Total protein was extracted after transfection and precipitated with myc antibody. The precipitate was examined by immunoblotting with either p38 antibody or myc antibody as indicated. (F) myc-NP60 was co-transfected with Flag-JNK or Flag-ERK into 293T cells. Total protein was extracted after transfection and precipitated with myc antibody. The precipitation was examined by immunoblotting with flag or myc antibodies as indicated.