Ab184637

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).

Briefly, paraffin-embedded sections of normal human skin samples were cut, then incubated for 30 min at 60°C, then washed with limonene 3 times and hydratated by immersing subsequently in 100%, 90%, 80%, 70%, and 50% ethanol solutions. Then samples were boiled for 10 min in 10 μM solution of sodium citrate (Sigma) and incubated in 0.1 M solution of sodium tetrahydroborate overnight at 4°C. Then samples were washed once with PBS and permeabilized with 0.30% Triton-X-100 in PBS for 30 min. Samples were blocked with 10% goat serum in PBS for 1 h and then exposed to primary antibodies. Samples were treated with anti-FOXM1 (Ab184637, Abcam, 1:20), anti-p63 (Ab735, Abcam, 1:50), anti-Keratin10 (PRB-159P, Covance; 1:1000), and anti-Keratin14 (PRB-155P, Covance; 1:1000) primary antibodies overnight at 4°C. Samples were washed 3 times with PBS and then treated with a 488- or 568-Alexa Fluor secondary antibodies (1/1000 dilution; Invitrogen) and DAPI for 1 hour. After three washes in 1X PBS, the slides were mounted using the Prolong Antifade kit (Invitrogen). Slides were analyzed with a confocal laser microscope (NIKON Eclipse Ti). Detection of the signal was performed using EZ C.1 software (Nikon).