Fig. 3.

p62 interacts with c-Jun. (A) HEK293T cells were transfected with GFP-p62 and pcDNA3.1 or Strep-FLAG (SF)-c-Jun plasmids using PEI transfection reagent. At 36 h post-transfection, cells were treated with MG132 (10 µM) for 12 h and lysed in RIPA buffer. SF-c-Jun was purified with anti-FLAG affinity gel. Cell lysates and purified samples were immunoblotted using the antibodies indicated. Note: MG132 was added to prevent the degradation of c-Jun upon p62 expression, which resulted in approximately equal c-Jun protein levels in two cell lysates. (B) HEK293T cells were transfected with FLAG or FLAG-p62 for 48 h. The resulting cell lysates were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates and cell lysates were subjected to immunoblot with the antibodies indicated. (C) UntransfectedHEK293T cells were treated with MG132 (10 µM) for 12 h and lysed in RIPA buffer. IgG or anti-c-Jun antibodies were used for immunoprecipitation of endogenous proteins. Endogenous proteins in cell lysates and the immunoprecipitates were immunoblotted with the antibodies indicated.