Proximity labeling with TurboID

Stable MDA-MB-231 cells expressing LHPP-TurboID, TurboID, and the empty pLVX vector 70–90% confluent in 10 cm plates were treated with 500 μM biotin for 10 min. After treatment, protein lysates were lysed in 1X RIPA buffer, as previously described. For each condition, 300 μg of cell lysate was incubated with 25 μL of streptavidin beads (ThermoFisher Scientific, 88817) and 500 μl of 1X RIPA overnight at 4°C with agitation. The protein enrichment was performed as previously described in (³⁴). The biotinylated proteins were eluted in 3X Laemmli buffer supplemented with 30 μL of 2 mM biotin and 20 mM DTT, boiled at 95°C for 10 min, and submitted to the Salk Institute Mass Spectrometry Core for protein identification using Tandem Mass Tag (TMT) labeling quantitative mass spectrometry.