Imagequant tl 1d software

Cell lysates were prepared in ice-cold whole-cell extract buffer (50 mM TRIS-HCl, pH 8.0, 4 M urea and 1% Triton X-100) supplemented with a complete protease inhibitor mixture (Roche Diagnostics, 04693132001). Aliquots (30 μg) of protein were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking non-specific binding for 1 h (5% non-fat milk in 0.1% TBS/T) at room temperature, the membranes were probed with antibodies as follows: from Abcam, Cambridge, UK: CDK2 (ab32147), GAPDH (ab128915), cyclin E1 (1655-1), p21 (3733-1), c-myc (ab32072), lamin A+C (ab108595); from Cell Signaling Technology, Danvers, MA, USA: cyclin B1 (12231), cyclin D1 (2978), E2F-1 (3742) and phospho-Rb (Ser807/811) (9308); from Santa Cruz Biotechnology, Dallas, TX, USA: cyclin A1 (B-8, sc-271682), and α-tubulin (sc-5286) and GADD45α (A11768, Abclonal Biotechnology), PUMA (55120-1-AP, Proteintech), p53 (bs-8687R, Bioss). After incubation at 4°C overnight, the membranes were incubated with the appropriate secondary antibodies (anti-mouse IgG(H+L) or anti-rabbit IgG(H+L), 1:2,500; SeraCare) for 1 h at room temperature. Protein bands were visualized using an ECL kit (Cat. 54-61-00, KPL). The results were visualized using an ImageQuant LAS 4000 Mini and processed using ImageQuant TL 1D software (General Electric Company).

TE1 cells were seeded on a 3.5-cm dish, treated with various compounds, and lysed in RIPA buffer. Proteins were separated on SDS polyacrylamide gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked and immunoblotted with primary antibodies at 4 °C overnight, followed by appropriate secondary antibodies. GAPDH was used as the loading control. Membranes were visualized under an Image Quant LAS 4000 mini and processed by Image Quant TL 1D software (General Electric Company).
The primary antibodies C-RAF (Cat.9422), MEK1/2 (Cat.9122), p-MEK (Ser217/221, Cat.9154), ERK (Cat.4695), p-ERK (Tyr202/Tyr204, Cat.4370), GAPDH (Cat.5174) and snail (Cat.3879) were purchased from Cell Signaling Technologies (Danvers, MA, USA). RAS (Cat.ab137739) and B-RAF (Cat.ab33899) were purchased from Abcam (Cambridge, UK).

Protein samples from co-immunoprecipitation were loaded onto SDS-polyacrylamide gels using SDS-sample buffer, separated by gel-electrophoresis and analysed by immunoblotting. All antibodies were diluted in 50 mM Tris [pH 7.2], 150 mM NaCl with 0.2% Tween-20 and 5% skim milk powder. Antibodies and dilutions are listed in Supplementary Table S1. For visualization, we used Amersham ECL Prime or Select Western Blotting Detection Reagent (GE Healthcare) in combination with the Fusion FX-6 Edge system (Vilber Lourmat). Quantification was performed in a semi-automated manner using the ImageQuant TL 1D software (GE Healthcare Life Sciences) with a rolling-ball background correction.