APEX2 proximity labeling

We followed an APEX2 protocol as described in prior publications (⁴⁷). Stably expressing ACKR3-APEX2 β-arrestin 1/2 KO cells were placed in a 6cm dish with starvation media (MEM, 1% P/S, and 0.5% FBS) for at least four hours prior to experimentation. 30 minutes to 1 hour prior to labeling, biotin phenol (Iris Biotech) in DMSO stored at −80°C was added to the cells to a final concentration of 500μM. Cells were stimulated for a total of 3 minutes with CXCL12 at a final concentration of 100nM. During the final minute of agonist simulation, a solution of hydrogen peroxide in PBS was added to a final concentration of 1mM. Next, the cell media containing biotin phenol, CXCL12, and hydrogen peroxide was aspirated and cells were washed with 3mL of fresh quenching buffer (10mM sodium ascorbate, 5mM Trolox, 10mM sodium azide) three times to stop the proximity labeling. Cells were then lysed using 500mL of radioimmunoprecipitation (RIPA) buffer (150mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate 0.1% sodium dodecyl sulfate, 50mM Tris pH 7.4) supplemented with cOmplete protease inhibitor (Sigma-Aldrich) and quenching reagents (10mM sodium azide, 10mM sodium ascorbate, and 5mM Trolox). Cells were collected, left to sit on ice for two minutes, and then rotated at 4°C for 1 hour, followed by subsequent centrifugation at 17000G for 10 minutes where the supernatant was then collected and stored for further analyses.