Figure 5. Proximity tagging with A-SDC1_45,47 to capture interactomes in live cells.

(A) Cartoon depicting proximity tagging with APEX2 fused SDC1_45,47HEP (A-SDC1_45,47HEP). After incubation of live cells with A-SDC1_45,47HEP, excess unbound protein is washed away and cells are incubated with biotin phenol (yellow circle, 0.5 mM, 30 min). The APEX2 peroxidase catalyzes the formation of short lived biotinyl radicals that react with proximal proteins upon addition of H₂O₂. (B) Ext1^−/− mESCs subjected to the proximity tagging protocol qualitatively demonstrate differences in interactomes (biotin, purple) based on membrane anchoring (+ cholPEGNTA) and glycosylation (A-SDC1_45,47HEP). Data representative of three biological replicates.