Protease inhibitor cocktail 100

Cultured cells were collected 24 h posttransfection and lysed using RIPA lysis and extraction buffer (ThermoFisher) to obtain whole cell lysates. Snap-frozen livers were thawed on ice, and 150 mg of each were weighed out into 2 ml of soft tissue homogenizing CK14 tubes (Bertin). Then, 1.5 ml of Triton X-100 lysis buffer (150 mM NaCl, 1.0% Triton X-100, 50 mM Tris) were added to each tube, and livers were homogenized using a Precellys 24 tissue homogenizer (Bertin) to obtain liver lysates. All lysis buffers were supplemented with protease inhibitors by diluting Protease Inhibitor Cocktail (100×) (Cell Signaling Technology) to 1× in the appropriate lysis buffer. Cell and liver lysates were then precleared with 100 µl of G-Sep Agarose CL-6B beads (G-Biosciences) for 1 h and subsequently incubated with 100 µl of Immobilized Biotin Resin (G-Biosciences) for 3 h at room temperature. Beads containing the immobilized protein were then washed and resuspended in a 50 µl solution containing TEV protease (New England Biolabs) and incubated overnight at 4 °C. The resulting bead solution was then filtered using 0.65 µm Ultrafree-MC centrifugal filters (Millipore Sigma) and the filtrate was run on LC-MS/MS.