BioID2_PHB2(V1)_PHB2(V3)-BirA-HA_HEK293

HEK293 cells that stably express PHB2 (V1 or V3) fused at C-terminus with the biotin ligase from A. aeolicus were incubated with 100 μM biotin for 18 h. The mitochondrial fractions were lysed in RIPA buffer, and the clarified supernatants were incubated with streptavidin agarose beads for 2 h at 4° C. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37° C. Three biological replicates of the samples were individually prepared and analyzed by LC-MS/MS. Label-free quantification was performed using Proteome Discoverer 2.2 software.