Extended Data Fig. 8: AND-gated proximity labeling.

a, Western blot analysis of K562^HER2+/EGFR+ treated with αHER2-SpyN (100 nM, eNrdJ-1^cage) and SpyC-αEGFR (100 nM) for 2 hr at 37 °C, followed by HA-tagged SpyTag003-APEX2 (300 μM) for 20 min. b, K562 (wildtype), K562^HER2+, K562^EGFR+, and K562^HER2+/EGFR+ cells were treated individually as described in panel a. Following washing, APEX2 proximity labeling was induced by the addition of biotin-phenol (250 μM) and H₂O₂ (1 mM). The reactions were quenched with sodium ascorbate (10 mM) and Trolox (5 mM) at room temperature after two minutes. Samples were then analyzed by Western blot using Strep-800 to detect biotinylation. c-e, Time-course study of APEX2 labeling. The cells were treated as in panel b with labeling quenched at the indicated time points. αHER2-SpyN/SpyC-αEGFR was used with the K562^HER2+/EGFR+ cells; αHER2-SpyN/SpyC-αEpCAM was used with the OE19 cells; αEGFR-SpyN/SpyC-αEpCAM was used with the A431 cells. SMART-SpyCatcher employed eNrdJ-1^cage in all cases. f, Displayed are the employed AND gates used in the following experiments (note, lettering differs from that in Fig. 3). Each AND gate constitute a pair of SpyN and SpyC protein fusions and/or synthetic ligand conjugates; for instance, AND gate D is generated by αCEACAM6-SpyN and SpyC-αHER2. g-j, AND-gated proximity labeling using the described AND gates, SpyTag003-APEX2 and the workflow described under panels a-e. In each case, the lane expected to give proximity labeling is indicated in red. When applicable, cells were treated for 12 hr beforehand with doxycycline (DOX, 200 ng/mL) to induce the expression of FLAG-CXCR4-eGFP (OE19^CXCR4DOX) or FLAG-ADORA2A-mCherry (OE19^A2ADOX). SpyC-Cys indicates the unconjugated variant. Asterisks indicate endogenously biotinylated proteins. Data shown in panels a-e, and g-j are representative of two and three independent experiments, respectively.