The new VIM consensus sequence is necessary and sufficient for p97 binding. A, streptavidin Sepharose pull-down assay (PD) using a biotinylated peptide spanning the VIM motif of ZNF744 (VIM peptide) and the indicated recombinant p97 variants (p97, p97ΔN, and p97ΔC). p97 binding to the immobilized peptide was analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue staining. B, yeast two-hybrid analysis. PJ69-4a yeast cells transformed with the indicated combinations of bait (pGAD-) and prey (pGBDU-) plasmids were spotted onto synthetic complete medium agar plates lacking uracil and leucine (control) or lacking uracil, leucine, and histidine (−His). Growth was monitored after 3 days. ZNF744R, R655A; ZNF744AA, A661L/A662L; ZNF744RL, R665A/L666A. C, interaction in human cells. Lysates (Input) of HEK293T cells ectopically expressing the indicated FLAG-tagged ZNF744 variants or UBXD1 as a positive control were subjected to immunoprecipitation using anti-FLAG antibody (IP: FLAG). Immunoprecipitated ZNF744, UBXD1, and co-precipitated endogenous p97 were detected by Western blot (WB) using anti-FLAG and anti-p97 antibodies, respectively.