*4.5. Western Blotting and Co-Immunoprecipitation (Co-IP)*

For Western blotting analysis, cells were washed twice with PBS and lysed on ice for 20 min by using a RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS), supplemented with protease and phosphatase inhibitors. Cellular extracts were further centrifuged for 30 min at 13,000 rpm at 4◦C. The pellet was removed, and the protein concentration in the supernatant (whole-cell lysate) was determined by the Bradford assay. The samples containing 30 µg of protein were resolved on 4% to 12% Bis-Tris or 3% to 8% Tris-acetate NuPAGE gels (Invitrogen, Carlsbad, CA, USA), transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), probed with specific antibody, and visualized by enhanced chemiluminescence (Western Lightning Plus-ECL reagent, Perkin Elmer, Waltham, MA, USA) on an gel-documenting system "Fusion Solo WL.2M" (Vilber Lourmat, Collégien, France). Densitometric analysis was performed in FIJI Software (Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI, USA).

For Co-IP, cells were washed with PBS and lysed on ice for 15 min using by TEB buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 and 10% glycerol), supplemented with protease and phosphatase inhibitors. The lysates were cleared by centrifugation and further incubated with the corresponding precipitating Abs overnight (rotating device at 4 ◦C). Then protein A and G Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to the samples and incubated for 1 h (rotating device at 4 ◦C). The beads were washed 3 times with TEB buffer and resolved by SDS-PAGE. Then 1X sample buffer was added to the resolved precipitates and the mixture was boiled for 5 min. Subsequently, Western blotting was performed as indicated above.
