APEX2 Proteomic Analysis

Our APEX2 protocol is based on the published protocol of the Ting laboratory (^{Hung et al., 2016}). Briefly, HEK293 cells were transfected with the indicated plasmids and allowed to express the proteins over a 24-hour period. Cells were then incubated with 500 μM biotin tyramide (Iris Biotech, cat 41994-02-9) in DMEM + 10% FBS for 1 hr, treated with 1 mM H₂O₂ for 1 min to induce the APEX reaction, treated with quenching solution (10 mM sodium ascorbate, 5 mM Trolox and 10 mM sodium azide, in DPBS) to end radical formation, washed 3x in PBS and either fixed in DPBS + 4% formaldehyde for 15 min (if the cells were to be imaged) or collected in DPBS + protease inhibitor (for affinity purification). Cells that were imaged were labeled using CF640-conjugated streptavidin to label biotinylated proteins (CF640R Streptavidin, Biotium cat 29041). Affinity purification was performed using streptavidin-conjugated magnetic beads (Pierce, REF 88817) utilizing the steps and buffers of the previously referenced protocol [13]. The affinity purified samples were then either submitted for mass spectrometry analysis or subjected to gel electrophoresis to generate the western blot figures found in the manuscript. Membranes were probed with mouse anti-GFP (Sigma, G6539) then labeled with LiCor IRDye 680LT Goat α-Mouse (926–68020) and LiCor IRDye 680RD Streptavidin (926–68079). Membranes were imaged on a LiCor Odyssey Classic system. LC MS/MS with label free quantitation was performed on purified samples in triplicate after a precipitation step to reduce detergent and free biotin concentration. A threshold of 2+ unique peptides identified was used and proteins that did not satisfy this requirement were not included in the analysis.