Fig. 3: Gβγ signaling is essential for CXCR4 signaling and PTM.
a Illustration of the current model of GPCR desensitization. Perturbations used to antagonize different components of the pathway are highlighted in red. b Representative western blot illustrating the effects of Gβγ inhibition (Gallein, 10 μM) treatment on CXCL12-induced AKT S473 and ERK1/2 phosphorylation. Cells were pretreated with Gallein for 30 min prior to and throughout each signaling time course. c, d Western blot quantification of AKT S473 and ERK1/2 phosphorylation after Gβγ inhibition. Relative signaling protein phosphorylation was calculated by dividing the phosphorylated protein detection by total signaling protein detection and then normalized to the 5 min time point of the control sample. e Representative western blot illustrating the effect of Gβγ inhibition (Gallein 10 μM) on CXCR4 PTM. Cells were pretreated with Gallein for 30 min prior to and throughout the signaling time course. f Western blot quantification of CXCR4 UMB2 detection (i.e., PTM) upon Gβγ inhibition. CXCR4 PTM was calculated by dividing UMB2 detection (non-PTM CXCR4) by MYC intensity (total CXCR4) and normalized to the 0 min control sample. For all experiments a minimum of three independent replicates were performed. All experiments were conducted in RPE cells overexpressing WT CXCR4 and stimulated with 12.5 nM CXCL12 for the stated time course. Individual data points from each experiment are plotted; mean, SD, and median line. Statistical significance *p < 0.05. Complete raw blots are shown in Supplementary Fig. 9.
