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. 2020 Jan 20;295(9):2866–2884. doi: 10.1074/jbc.RA119.010077

Figure 8.

Figure 8.

A, entry of FAM-9R–conjugated peptides into A549 cells. Peptides were applied to A549 cells at a final concentration of 1 μm, and the cells were monitored by confocal microscopy at a variety of time points. Representative images are shown and labeled accordingly. B, inhibition of Cdc42–effector interactions by C1 and second generation peptides (P1 and P7). The ability of Cdc42 to interact with its effector WASP in the A549 lysate was monitored by GST–WASP pulldown experiments. GST–WASP GBD immobilized on GSH-agarose beads was used to pull down endogenous Cdc42 from A549 lysates in the presence of various peptide concentrations. The levels of Cdc42 bound were analyzed by Western blotting. Representative Western blottings from unconjugated C1, P1, and P7 treatments are shown in the upper panels. Densitometry was performed on the results from replicate experiments (n ≥ 3), and the amount of Cdc42 present in pulldowns, expressed as a percentage of levels in the absence of peptides, are plotted in the lower panel. Data for FAM-9R–conjugated peptides are also included. C1, pink; FAM-C1–9R, red; P1, green; P7, cyan; FAM-P7–9R, purple. Approximate IC50 values were calculated from the curve fits: C1, 190 ± 62 nm; FAM-C1–9R, 2200 ± 740 nm; P1, 18 ± 3.5 nm; P7, 1.2 ± 0.28 nm; FAM-P7–9R, 65 ± 12 nm. C, target engagement by peptides. CETSA experiments were run in A549 lysate at 56 °C treated with varying concentrations of FAM-P7–9R peptide to assess target engagement. Cdc42 and actin (as a control) levels were quantified by Western blotting, and a representative blot is presented showing destabilization of Cdc42 with increasing peptide concentrations. n = 3. D, target engagement after cell penetration. Whole-cell CETSA experiments were run in A549 cells at 51 °C after 4 h of incubation with varying concentrations of FAM-P7–9R peptide to assess target engagement after external application of peptide. After lysis, Cdc42 and actin levels were quantified by Western blotting, and a representative blot is presented showing destabilization of Cdc42 with increasing peptide concentrations. n = 3.