HEK293T cells (1 × 107) were seeded into 15-cm tissue cultures plates for 24 hours, and then, DNA constructs encoding Halo or SNAP tagged genes of interest were transfected using Lipofectamine LTX (Thermo Fisher Scientific). Forty-eight hours after transfection, cells were harvested and washed twice with ice-cold PBS. The ice-cold PBS washed cells were resuspended in 300 μl of mammalian cell lysis buffer (Promega) containing 50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1 mM benzamidine HCl, 55 μM phenanthroline, 1 mM PMSF, 10 μM bestatin, 5 μM pepstatin A, and 20 μM leupeptin. Next, the cells were raptured by passing through a 26-gauge needle five to seven times, followed by centrifugation at 21,000g for 30 min at 4°C. The resulting 300 μl of cell extracts were collected into a new tube and diluted with 700 μl of tris-buffered saline [50 mM tris-HCl (pH 7.4), 137 mM NaCl, and 2.7 mM KCl]. To remove insoluble materials, the diluted cell extracts were further centrifuged at 21,000g for 10 min at 4°C. Next, 1000 μl of cell extracts were incubated with magnetic beads prepared from 100 μl of Magne HaloTag slurry for overnight at 4°C. Beads were washed four times (750 μl of buffer per wash) with wash buffer [50 mM tris-HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.05% NP-40] before elution. Proteins were eluted using elution buffer containing 50 mM tris-HCl (pH 8.0), 0.5 mM EDTA, 0.005 mM dithiothreitol (DTT), and 2 U of AcTEV Protease (Thermo Fisher Scientific) for 2 hours at room temperature. The eluate was further passed through a Micro Bio-Spin column (Bio-Rad, Hercules, CA) to remove any residual particles of beads before proteomic analysis.

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