Fig. 1: CXCL12-depedent AKT S473 phosphorylation and CXCR4 PTM are independent of CXCR4 localization.

a Illustration of CXCR4 mutant receptor constructs. The gray box denotes the binding region of the UMB2 antibody that is sensitive to CXCR4 PTM. b Flow cytometry analysis of overexpressed WT and mutant receptor plasma membrane localization in RPE cells. Data were normalized to total receptor and WT plasma membrane expression. c Flow cytometry analysis of WT and mutant receptor total expression. Individual data points were normalized to WT CXCR4 expression. d Representative microscopy images illustrating the distribution of WT and K3R CXCR4 localization within RPE cells overexpressing each construct. CXCR4 was labeled with a FLAG antibody and the Golgi was detected using a GM130 antibody. Scale bar is 10 μm. Images were captured using 60× magnification on a spinning disk confocal microscope. e Representative western blot illustrating CXCL12-induced (12.5 nM) AKT S473 phosphorylation and CXCR4 PTM for WT and mutant receptors. Total CXCR4 was detected using a MYC antibody and unmodified CXCR4 by UMB2. f Western blot quantification of AKT S473 phosphorylation for WT and mutant CXCR4. Relative AKT phosphorylation was calculated by normalizing phospho-AKT to total AKT band intensity, and secondly to the 5 min control time point. g Western blot quantification of CXCR4 PTM (i.e., UMB2 detection). A decrease in UMB2 detection is correlated to increased CXCR4 PTM. CXCR4 PTM was calculated by dividing the UMB2 intensity by the MYC intensity (total CXCR4) and secondly to the 0 min time point for the WT receptor. h Flow cytometry analysis of agonist-dependent WT and mutant CXCR4 PTM. Relative UMB2 detection was determined by dividing median UMB2 detection by total CXCR4 fluorescence and normalized to 0 min WT CXCR4. All experiments were conducted a minimum of three times in RPE cells overexpressing WT or mutant CXCR4. Individual data points from each experiment are plotted; mean, standard deviation (SD), and median line. Statistical significance *p < 0.05. Example of flow cytometry gating strategy is shown in Supplementary Fig. 6. Complete raw blots are shown in Supplementary Fig. 7.