Complete mini edta free protease and phosstop phosphatase inhibitor cocktails

At 48 h post-transfection, cells were dissociated from the plate surface with 1× phosphate-buffered saline (PBS) containing 10 mM EDTA, subsequently washed with cold 1× PBS, and lysed in IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5% Nonidet P40 substitute (NP-40; Solarbio, Beijing, China) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor cocktails (Roche, Bransburg, NJ, USA). Cells were lysed on ice for 30 min then cleared by centrifugation at 17,000× g for 10 min at 4 °C. After centrifugation, the supernatant was incubated with 30 μL Strep-Tactin Sepharose beads (IBA Lifesciences, Göttingen, Germany) diluted in IP Buffer for 2 h. Beads were then washed three times with 1 mL IP buffer supplemented with 0.05% NP-40 and transferred to a new tube with a final wash in 1 mL IP buffer. Proteins were eluted by agitating beads in 40 μL IP buffer supplemented with 2.5 mM D-desthiobiotin (IBA Lifesciences) on a vortex mixer at room temperature for 30 min. We reserved 10% of each eluate for western blotting and silver staining. The remaining eluate was removed for mass spectrometry (MS).