Anti plk2

Whole cell lysates were extracted using a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X) with an EDTA-free protease inhibitor cocktail (Bio Basics Inc.) 20 ug of total protein was used to perform Western blot analysis. Primary antibodies were purchased accordingly, anti-PLK2, anti-PLK3, and anti-DNMT3b (from Santa Cruz), anti-PLK1, anti-PLK4, anti-GAPDH, and anti-DNMT3A (from Cell Signalling), and anti-DNMT1 and anti-Actin (from Sigma). For secondary antibodies, anti-rabbit (from Cell Signalling) and anti-mouse HRP (from Sigma) were used. Bands were visualized by ECL (Thermo Scientific) and blots were acquired on an Alpha Innotech Multimage™ Light Cabinet and densitometry analysis was carried out using OptiQuant software Version 5.0.

Whole-cell lysates were prepared by using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Roche, Nutley, USA), and proteins were quantified by using a bicinchoninic acid (BCA) protein quantitation kit (Thermo Scientific). Ten micrograms of whole-cell lysate per lane was used for immunoblotting. The following antibodies were used: anti-KRAS (Sigma-Aldrich, St. Louis, USA), anti-DCBLD2 (Cell Signaling, Danvers, USA), anti-p-AKT-Ser473 (Cell Signaling), anti-p-AKT-Thr308 (Cell Signaling), anti-total-AKT (Cell Signaling), anti-PIK3R2 (Santa Cruz, Dallas, USA), anti-PLK2 (Santa Cruz), and anti-UBQLN2 (Millipore, Billerica, USA) at 1:1000 dilution; anti-β-Actin and anti-GAPDH (Cell Signaling) at a dilution of 1:2000.