FIGURE 6.

FITC-SP1 can enter mammalian cells and inhibits the interaction between RalB and Sec5. Shown are wide-field (A) and single-cell images (B) of HEK293T cells treated with 10 μm FITC-Peptide 1, 10 μm FITC-SP1, or 10 μm FITC control overnight. Chemical stapling enables FITC-SP1 to penetrate the cell membrane and localize in both the cytosol and nucleus. C, SP1 disrupted RalB-Sec5 interaction in cells. HEK293T cells were treated for 24 h with SP1 and SP6 as indicated. The numbers above the Western blot represent the relative intensities of the Sec5 band, normalized to the no-peptide control. Endogenous RalB was immunoprecipitated (IP) with mouse anti-RalB antibody. Immunoblotting was conducted with primary antibodies against RalB and Sec5. * marks the band specific to Sec5. Ab, mouse anti-RalB antibody alone. WCL, whole cell lysate.