- Grow NIH/3T3 cells (ATCC, CRL-1658) in DMEM+GlutaMAX (Gibco, 31966-021, Lot 1852045) supplemented with fetal bovine serum (Gibco 270-106) and Pen/Strep (Gibco 15140), at 37°C and 5% CO2 to 70% confluency.
- Transfect cells with a plasmid encoding mVenus-tagged p27K- (kindly provided by Toshio Kitamura, University of Tokyo; Oki et al., 2014) using EndofectinMax (GeneCopoeia) following the manufacturer's instructions.
- Serum starve cells for 48h. Use untransfected NIH/3T3 cells starved for 48h as control.
- Lyse cells in RIPA buffer (10mM PBS pH7.2, 2mM EDTA, 1% NP-40, 1% Triton X-100, protease inhibitors) at 4°C.
- Denature total cellular lysates proteins in 1x SDS-sample buffer and separate proteins on a freshly cast denaturing 10% SDS-PAGE (Laemmli, 1970).
- Transfer proteins onto 0.2µm Protran membrane (GE Healthcare, 10600004, A10043108) with a Western blotting system for 1h at 400A (Towbin et al., 1979).
- Block the membrane in TBST (50mM Tris-HCl, pH7.4, 150mM NaCl, 0.2% Tween 20) containing 5% milk (blocking solution) for 60min at RT.
- Incubate membrane with primary mouse anti-P27 antibody (antibodies-online, ABIN3025539, lot V2438-171009) diluted 1:250 in blocking solution ON at 4°C.
- Incubate membrane with secondary anti-mouse IgG (whole molecule), HRP-linked (Sigma-Aldrich, A9044, lot 034M4761) diluted 1:5000 in TBST for 45min at RT.
- Wash membrane with TBST for 30-45min at RT.
- Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062); image capture via Chemidoc Imaging System (BioRad).