Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket.

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet: Figure 5. Targeting Rho- and Rab-family GTPases (A) Covalent modification of Rac1(G12C) with compounds 1–10 (50 mM, 12 h). (B) Covalent modification of RhoA(G14C) with compounds 1–10 (50 mM, 12 h). (C) Time-dependent covalent modification of Rac1(G12C), RhoA(G14C), and Rac1(WT) with divarasib (50 mM). (D) Time-dependent covalent modification of various Rac1 mutants with divarasib (50 mM). (E) Covalent modification of Rab1A(S20C) with compounds 1–10 (50 mM, 12 h). (F) Covalent modification of Rab5C(S30C) with compounds 1–10 (50 mM, 12 h). (G) Time-dependent covalent modification of Rab1A(S20C), Ypt1(S17C), Rab5C(S30C), and RabL5(WT) with MRTX1257 (50 mM). (H) Time-dependent covalent modification of various Rab1A mutants with MRTX1257 (50 mM). (I) Peptides showing significant differences in HDX at any time point (>0.35 Da and >4.5%) mapped onto a homology model of Rab1A based on adagrasib-bound K-Ras(G12C) (PDB: 6USZ). (J) Differential scanning fluorimetry of Rac1(G12C, K96H),GDP and Rac1(G12C, K96H),GDP,divarasib adduct. (K) Rac1 activity measured by Rac1 G-LISA. HeLa cells were transiently transfected, treated with different concentrations of divarasib for 12 h, and lysates were tested at 0.5 mg/mL. Data are presented as mean ± SEM (n = 2) and are representative of three independent experiments. See also Figures S2, S3, and S4.

Article Snippet: Rac1 downstream signaling assays The effects of Rac1 inhibition on downstream signaling was assessed on COS7 cells via the Active Rac1 Detection Kit from Cell Signaling Technology (CST) 8818S, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Activity Assay, Transfection

Journal: Journal of Lipid Research

Article Title: Oleate activates PLD2 lipase and GEF activity by modulating membrane microdomain dynamics via S-acylation

doi: 10.1016/j.jlr.2025.100939

Figure Lengend Snippet: PLD2 functions as a GEF for Cdc42 activation. A: Confocal microscopy images of HEK293T cells overexpressing EGFP-PLD2 (WT, K758R, ΔCRIB). Cells were stained with Phalloidin-TRITC to visualize filopodia-like cell protrusions. The scale bar in the first micrograph applies to all images in the series. The zoomed-in channels correspond to the regions highlighted by the yellow rectangles in the merged image. B, C: Western blot analysis of GTP-Cdc42 (active form) and total Cdc42 (input) in MDA-MB-231 cells (B) and HEK293T cells (C). MDA-MB-231 cells were treated with BSA (Ctrl)/100 μM OA for 10 min or with DMSO (Ctrl)/100 μM MβCD for 3 h HEK293T cells expressing EGFP-vector/EGFP-PLD2 (WT/ΔCRIB/K758R) were lysed to detect basal level of Cdc42 by Western blot. HEK293T cells expressing PLD2-WT/K758R/C223AC224A were treated with BSA (Ctrl)/100 μM OA for 10 min. Data are presented as mean ± SEM (3 independent experiments). Statistical significance was determined by unpaired two-tailed Student's t-tests with their corresponding control groups (B and right panel in C) or one-way ANOVA followed by Dunnett’s multiple comparisons test to WT (left panel in C). Significance thresholds were defined as: ns, not significant, P ≥ 0.05; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001.

Article Snippet: Cdc42 activation was measured using the Cdc42 Activation Assay Kit (Cell Signaling #8819) according to the manufacturer’s instructions.

Techniques: Activation Assay, Confocal Microscopy, Staining, Western Blot, Expressing, Plasmid Preparation, Two Tailed Test, Control