Figure 6.

NW peptide interacts with intracellular prohibitins. (A) Schematic illustration of the peptide–Fc fusion construct. (B) The peptide–Fc fusions (NW–Fc and CtlP–Fc) were expressed in HEK293T cells, purified as described in the Section 4, and then analyzed by SDS-PAGE, followed by Coomassie blue staining. Duplicate gels were transferred to nitrocellulose membranes and probed with either anti-human IgG Fc monoclonal antibody (C) or anti-PHB1 or anti-PHB2 monoclonal antibodies (D). NT proteins were prepared from non-transfected cells. (E) Replacement of W with A at positions 2, 6, and 12 (NW-A) inhibited the binding of the NW peptide to prohibitins. Whole-cell protein lysates were incubated with either the wild-type NW peptide (NWYLPWLGTNDW), the alanine-substituted NW peptide (NAYLPALGTNDA), or the control peptide (FYPSYHSTPQRP) and then processed as described for Figure 4A,B. CtlP, control peptide.