Horseradish peroxidase conjugated secondary antibody rabbit and mouse

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).

Cells were lysed in SDS lysis buffer (100 mM Tris рН 8.8, 1% SDS, 5 mM EDTA, 20 mM DTT, and 2 mM AEBSF). Total protein extracts were resolved in SDS polyacrylamide gel and blotted onto a Hybond PVDF membrane (GE Healthcare, Chicago, IL, USA). 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-IRS1 (D32G12, Cell Signaling), anti-p63 (clone 4A4, Santa Cruz Biotechnology), anti-p-IRS1 (Ser612, clone C15H5, Cell Signaling), anti-p-AKT (Ser473, clone D9E, Cell Signaling), anti-AKT (clone #9272, Cell Signaling), anti-p-S6 Ribosomal Protein (Ser235/236, clone #2211, Cell Signaling), anti-S6 (Clone 5G10, Cell Signaling), anti-p44/42 MAPK (p-ERK1/2) (Thr202/Tyr204, Clone E10, Cell Signaling), anti-ERK1/2 (Clone 137F5, Cell Signaling).

Immunoblot analysis was performed using whole-cell extracts obtained by lysing the cell pellet with Triton buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 50 mM NaF, 1 mM EDTA 1 pH 8, 0.1% Triton) supplemented with proteases and phosphatases inhibitors. Proteins were resolved on an SDS-10% polyacrylamide gel and blotted onto a Hybond P PVDF membrane (G & E Healthcare). Membranes were blocked with PBST 5% non-fat dry milk, incubated with primary antibodies for 2 h at room temperature, washed and hybridized for 1 h at room temperature using the appropriate horseradish peroxidase-conjugated secondary antibody (rabbit and mouse; BioRad, Hercules, CA, USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, MA, USA). The following antibodies were used: anti- β actin (Sigma AC15, dilution 1:50000), anti-SETDB1 (Thermo Scientific 5H6D4, diluition 1:1000), anti-FLAG rabbit (Sigma F7425, diluition 1:1000), anti-HA (Abcam ab130275, diluition 1:1000), anti-p63 BC4A4 mouse (Abcam ab735, diluition 1:200), anti-p63 rabbit (Abcam ab97865, diluition 1:200), anti-p63 4A4 mouse (Sigma P3737, diluition 1:500), anti-p63 3.1 mouse [54 (link), 63 (link)] (diluition 1:500), anti-p53 (Santa Cruz, diluition 1:1000), anti-H3K9me3 (Millipore, diluition 1:1000). Supplementary Figures S5–S10 show un-cropped images of western blots.