Figure 3

P61A6 inhibits protein geranylgeranylation and activation of RhoA. A. Effects of P61A6 on geranylgeranylation of RhoA. H358 cells were treated with DMSO or increasing concentrations of P61A6 for 48 h. Whole cell lysates were prepared and analyzed for RhoA (upper panel) and actin (lower panel). B. Appearance of unprenylated form of Rap1 by treating the cells with P61A6. Rap1 is normally geranylgeranylated but the P61A6 treatment produces unprenylated Rap1 and this is detected by using an antibody specific for unprenylated Rap1. C. P61A6 causes an increase in cytosolic RhoA. H358 cells were treated with DMSO or P61A6 for 48 h. Cytosolic (C) and membrane (M) fractions were prepared and processed for SDS-PAGE, followed by Western blotting. Upper panel: RhoA; lower panel: cytosol marker RhoGDI. D. P61A6 inhibits RhoA activation in H358 cells. Cells were serum-starved in the presence of DMSO or P61A6 for 24 h. Then, cells were stimulated with 10% FBS in DMEM in the presence of DMSO or P61A6 for 30 min. Whole cell lysates were collected using Mg²⁺-containing lysis buffer, and GTP-RhoA was pulled down using GST-tagged Rhotekin-RBD protein beads (Cytoskeleton) following the manufacturer’s instructions. Whole cell lysates (inputs) and pull-down were resolved on SDS-PAGE for immunoblotting analysis using RhoA antibodies to detect total RhoA (bottom panel) and GTP-bound-RhoA (top panel), respectively.