Analysis of protein expression by western blotting

Sample buffer (150 µl; 5% glycerol, 50 mM Tris-HCl pH 6.8, 50 mM DTT, 1% SDS, 0.7 mM Bromophenol Blue) was used to lyse cells from a well of a 24-well plate. Proteins were separated on 10% gels prior to semi-dry transfer onto polyvinylidene fluoride (PVDF) membrane (ThermoFisher) for 60 min at 20 V using 48 mM Tris-HCl, 39 mM glycine, 20% methanol and 0.0375% SDS as the transfer buffer. Membranes were blocked using PBS with 0.05% Tween-20 and 5% milk (PBSTM) for 1 h (3% BSA was used instead of milk for blots with the anti-pTyr PY20 antibody), then incubated with primary antibody: affinity purified anti-Cog4 (1:500, Miller et al., 2013), anti-tubulin (1:2000, gift from M. Gerard Waters, Princeton University, Princeton, NJ), anti-ALP (1:6000, Santa Cruz Biotechnology sc-137213), anti-active β-catenin (1:2000, Millipore 050665), anti-phosphotyrosine (1:1000, with 3% BSA instead of milk, BD Bioscience clone PY20), anti-Akt (1:1000, Santa Cruz Biotechnology sc-1618), anti Akt-pThr308 (1:1000, Cell Signaling 13038), anti-Akt-pSer473 (1:1000, New England Biolabs 4060P) anti-pSmad2/3 (1:500, Santa Cruz Biotechnology sc-11769), anti-pERK (1:4000, R&D Systems AF1018) in PBSTM overnight at 4°C. Following six 10 min washes with PBSTM, appropriate HRP-linked secondary antibody (1:3000, Bio-Rad) in PBSTM was added for 1 h, then the blot was washed, and imaged on a GeneGenius Chemi-imager (Syngene) after application of Immobilon HRP substrate (Millipore). Quantification was carried out using ImageJ software.