Knockdown of GNB isoforms significantly impairs RAC1-mediated downstream signaling. (A) Representative western blots of scrambled or shRNA-transduced HL60 cells, which were left untreated or stimulated with IL-8 (100 ng/mL), lysed, and used to pull down GTP-bound active RAC1. Total RAC1 served as loading control (n = 3 experiments). (B) Number of adherent cells per field of view in the adhesion flow chamber after pre-incubation with TAT-WT RAC or (C) TAT-CA RAC construct (n = 4 experiments). *P < .05 vs all other groups. (D-E) Chemotactic migration of HL60 cells transduced with a scrambled construct or different shRNAs (n = 3 experiments; >60 cells). *P < .05 vs all groups. (D) Migration velocity and (E) accumulated (accum.) distance toward the applied IL-8 gradient in the ibidi μ-Slide. (F) Transduced HL60 cells were left unstimulated or incubated with IL-8 (100 ng/mL, 3 minutes, 37°C), fixed, and permeabilized to stain intracellular p-p38 MAPK. Staining was analyzed by using flow cytometry (n = 3 experiments). *P < .05 vs all other groups.