Figure 4.

TRAF6 and TAB1 mediate the up-regulation of ERK1/2 phosphorylation and MCL-1 expression by GADD34. A, HepG2 and SMMC-7721 cells were transfected with the EGFP-tagged GADD34 expression plasmid or EGFP vector and transfected with siControl or siTRAF6, followed by Western blotting analysis of MCL-1, p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), ERK1/2, TRAF6, and GADD34. B, HepG2 and SMMC-7721 cells were transfected with siControl or siTRAF6 and treated with or without 5 μm of MG132 for 24 h, followed by Western blotting analysis of MCL-1 and TRAF6. C, HepG2 cells were transfected with or without EGFP-tagged GADD34 and GFP-tagged TRAF6 expression plasmid, followed by immunoprecipitation with anti-TAB1 antibody and Western blotting analysis of TAB1 and TRAF6. The levels of GADD34–EGFP, TRAF6–GFP, and TAB1 in whole cell lysates were also detected by Western blotting. D, HepG2 cells were transfected with the EGFP-tagged GADD34 expression plasmid or EGFP vector, followed by immunoprecipitation with anti-TAB1 antibody and Western blotting analysis of TAB1 polyubiquitination (Poly-Ub). The levels of TAB1 and GADD34–EGFP in whole cell lysates were detected by Western blotting. E, HepG2 and SMMC-7721 cells were transfected with the EGFP-tagged GADD34 expression plasmid or EGFP vector and transfected with siControl or siTAB1, followed by Western blotting analysis of MCL-1, p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), ERK1/2, TAB1, and GADD34. IB, immunoblotting; IP, immunoprecipitation.