Figure 5.

Cigarette smoke extract (CSE) and cadmium alter Rac2 (Ras-related C3 botulinum toxin substrate 2) isoprenylation. (A) Schematic diagram of full-length Rac2_WT construct showing location of cysteine residues and Rac2 construct with Cys (C)→Ser (S) mutation at amino acid 189. (B) MH-S cells expressing empty or Flag-Rac2_WT were treated with vehicle, CdCl₂ (80 μM, 3 h), or CSE and were separated into aqueous or detergent fractions. Immunoblot analysis was performed. (C) MH-S cells expressing empty or Flag-Rac2_C189S were treated with vehicle, CdCl₂, or CSE and were separated into aqueous or detergent fractions. Immunoblot analysis was performed. (D) Macrophages expressing empty, Flag-Rac2_WT, or Flag-Rac2_C189S were treated with vehicle, CdCl₂, or CSE. Immunoblot analysis was performed in MH-S cells in isolated membrane fraction. MH-S cells coexpressing empty and YFP-Rac2_CA, or Flag-Rac2_C189S, or YFP-Rac2_CA and Flag-Rac2_C189S were treated with vehicle or CdCl₂. Quantification of immunoblot analysis was performed in isolated aqueous or detergent fractions for (E) GFP-Rac2 (n = 3) and (F) Flag-Rac2 (n = 3). (G) Histidine (His) pull-down in MH-S cells coexpressing empty or p67^phox-V5-His together with Flag-Rac2_WT or Flag-Rac2_C189S treated with vehicle or CdCl₂. ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA with Tukey statistical analysis was used for E and F. Results were repeated three times for all data in B–D and G, with representative immunoblots shown. A = aqueous; D = detergent; IB = immunoblot; PD = pull-down; V = vehicle; WT = wild type.