Fig. 3.

Characterization of the interaction between VKOR and TMX. (A) FLAG-tagged wild-type TMX or TMX mutants that carry mutations in its CXXC motif (CXXA or AXXA) were coexpressed with Myc-tagged wild-type VKOR or VKOR mutants in which the loop cysteines were changed to alanines (C43A or C51A). Controls were performed with expression of only one or neither (mock) of the two partners. The cells were treated with NEM and lysates subjected to denaturing immunoprecipitation (IP) with either FLAG or Myc antibodies. Following nonreducing SDS-PAGE, the samples were analyzed by immunoblotting with FLAG or Myc antibodies. The stars indicate the positions of adducts between TMX and VKOR. The plus sign indicates a dimer of the TMX AXXA mutant, which is seen even when it is expressed on its own. (B) The interaction of wild-type VKOR with a TMX mutant, in which the proline at the second position of the CXXC motif was altered (CPXA to CGXA), was analyzed as in A.