Anti keratin 10 prb 159p

Cells were lysed with SDS lysis buffer (100 mM Tris, pH 8.8, 1% SDS, 5 mM EDTA, 20 mM DTT, and 2 mM AESBF). Total cell extracts were resolved on an SDS polyacrylamide gel using the Mini-PROTEAN Tetra cell System (Bio-Rad) and blotted onto a Hybond P PVDF membrane (GE Healthcare) using the Mini Gel Tank transfer system (Life Technologies). After being blocked with PBST 5% non-fat dry milk (Bio-Rad), membranes were incubated over night with primary antibodies at +4°C, washed and hybridized for 1 h at room temperature using the appropriate horseradish peroxidase-conjugated secondary antibody (rabbit and mouse, Bio-Rad, Hercules, California, USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, Massachusetts, USA). The following antibodies were used: anti-FOXM1 (Ab184637, Abcam; dilution 1:300), anti-p63 (Ab735, Abcam; dilution 1:300), anti–β-actin (Sigma; dilution 1:30000), anti-p16 (SC-56330 (JC8), Santa Cruz Biotechnology; dilution 1:1000), anti-Rb (BD554136, BD Biosciences; dilution 1:500), anti-Keratin10 (PRB-159P, Covance; dilution 1:1000), anti-HA (16612, Covance; dilution 1:1000), anti-cMyc (SC-40 (9E10), Santa Cruz Biotechnology; dilution 1:200).