Figure 6.

The mZIP4 protein forms homodimers. A, HEK293 cells were transfected with the pcDNA3.1 empty vector or the same vector harboring the WT or ΔNED mutant mZIP4 tagged with HA, and the lysates were analyzed by immunoblotting with (+) or without (−) DTT. Arrows indicate dimeric species of WT and ΔNED mutant mZIP4-HA, respectively. B, Western blot analysis of WT and ΔNED mutant mZIP4-HA in the presence and absence of the cross-linking agent EGS. Lysates of HEK/WT-mZIP4-HA and HEK/ΔNED-mZIP4-HA cells were incubated for 30 min at room temperature with EGS. Reactions were quenched with 45 mm Tris-HCl (pH 7.5), followed by incubation at room temperature for an additional 30 min. The cross-linked products were analyzed by SDS-PAGE and immunoblotting with anti-HA antibody. Polypeptide species are consistent with the expected sizes of monomeric and dimeric complexes of WT and ΔNED mutant mZIP4-HA. Arrows indicate the dimeric species of WT and ΔNED mutant mZIP4-HA, respectively. C and D, HEK293 cells were transiently transfected with expression plasmids for mZIP4-HA and/or mZIP4-FLAG followed by immunoprecipitation with the indicated antibodies. Immunoblotting was performed with either anti-FLAG or anti-HA antibodies. Arrowheads indicate monomeric mouse ZIP4. The IgG chain is indicated.