Trib family proteins interact with MAPKKs in vivo. (A) HeLa cells were transiently transfected with FLAG-tagged Trib1 and HA-tagged MEK1 or MKK4. The cell lysates were immunoprecipitated with an anti-FLAG monoclonal antibody and immunoblotted with a rabbit anti-HA antibody (left) or immunoprecipitated with an anti-HA followed by immnunoblotting with anti-FLAG (right). The expression level of each protein was assessed by immunoblotting whole cell lysates (WCL) with anti-HA or anti-FLAG antibodies. (B) HeLa cells were transiently transfected with FLAG-tagged Trib1, Trib2 or Trib3 with HA-tagged MEK1. The cell lysates were immunoblotted with an anti-HA polyclonal antibody. The expression level of each protein was assessed by immunoblotting whole cell lysates with anti-HA or anti-FLAG antibodies. (C) Colocalization of MEK1 and Trib1. Trib1 and MEK1 were detected by immunofluorescence using an anti-FLAG antibody followed by a FITC-labeled secondary antibody or an anti-HA antibody followed by a TRITC-labeled secondary antibody. i, HA; ii, FLAG; iii, DNA; iv, Merge. (D) Coimmunoprecipitation between Trib1 and endogenous MEK1 in HeLa cells. The cell lysates were immunoprecipitated with an anti-FLAG monoclonal antibody and immunoblotted with a rabbit anti-MEK1/2 antibody. The expression level of each protein was assessed by immunoblotting whole cell lysates (WCL) with anti-MEK1/2 or anti-FLAG antibodies.