The thick-layer 3D cultures of ReN cells were homogenized with TBS extraction buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM NaVO3, 1 mM NaF, a protease inhibitor mixture (Roche Molecular Biochemicals), a phosphatase inhibitor cocktail (Thermo Scientific,), 2 mM PNT (EMD Millipore) and 1 mM Phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich) by using battery-operated spinning homogenizer (MIDSCI, St. Louis, MO, USA). After incubation on ice for 10 minutes, the samples were centrifuged for 1 hour at 100,000 × g to get TBS-soluble fractions. The TBS-insoluble pellets were then resuspended in 2% SDS extraction buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 2% SDS, 1% Triton X-100, 1 mM NaVO3, 1 mM NaF, a protease inhibitor mixture (Roche Molecular Biochemicals), a phosphatase inhibitor cocktail (Thermo Scientific), 2 mM PNT (EMD Millipore) and 1 mM PMSF (Sigma-Aldrich) and incubated on ice for additional 30 minutes. The samples were centrifuged for 1 hour at 100,000 × g and the supernatant fractions were collected as TBS-insoluble/2% SDS-soluble fractions. The 2% SDS-insoluble pellets were briefly washed with SDS extraction buffer and
