Imagequant las 4000 mini instrument

Equal amounts of proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked, primary antibody added, and incubated with secondary antibodies. Signals were detected using an Image Quant LAS 4000 Mini instrument (GE Healthcare, Wauwatosa, WI).

The cells for immunoblotting were prepared as we described previously (22 (link)). Briefly, Cells were solubilized in lysis buffer (1.0% NP-40, 50 mM HEPES, pH7.4, 150 mM NaCl), containing protease and phosphatase inhibitor (Thermo fisher scientific). Cell lysates were separated by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE), transferred to a Polyvinylidene difluoride (PVDF) membrane (Merck), and detected with the following antibodies using ECL substrate (Bio-Rad): rabbit anti-CCAAT enhancer binding protein (C/EBP)α anti-phospho-C/EBPα (Ser21), anti-phospho- C/EBPα (Thr221/226), anti-Erk1/2, anti-phospho- Erk1/2, anti-JNK, anti-phospho-JNK, anti-NF-κB(p65), anti-phospho-NF-κB(p65), mouse anti-IκBα (Cell Signaling Technology), mouse anti-IκBβ (Santa Cruz Biotechnology), mouse anti-βactin (Sigma-aldrich), horseradish peroxidase (HRP)-conjugated mouse IgG (Cell Signaling Technology), or rabbit IgG antibodies (Cell Signaling Technology). Digital images were obtained using an ImageQuant LAS4000 mini instrument (GE Healthcare). Densitometry was performed on scanned blots using the ImageQuant TL software program (GE Healthcare).

After cell-cycle arrest, 1 × 10⁶ cells were replated in DMEM with 10% FBS with or without PD0325901 (1 or 10 µM) or 0.1% DMSO in a 60-mm dish, and incubated for 48 h. The cells were obtained using radioimmunoprecipitation assay buffer (RIPA buffer; Thermo Fisher Scientific K.K., Kanagawa, Japan) and protease inhibitors (Roche Diagnostics, Basel, Switzerland). A BCA Protein Assay Kit (Thermo Fisher Scientific K.K.) was used to ascertain the protein quantities in the supernatant. Western blotting was performed as described previously^{27 (link)}. The following primary antibodies were incubated at 4 °C overnight: anti-PCNA (1:1000; Sigma-Aldrich), anti-p27 (1:1000; Cell Signaling Technology), anti-cyclin D1 (1:1000; Cell Signaling Technology), and anti-β-tubulin (1:1000; FUJIFILM Wako Pure Chemical). Horseradish peroxidase-conjugated secondary antibody (H goat anti-rabbit or anti-mouse IgG; 1:5000; Thermo Fisher Scientific) was incubated for 1 h at room temperature. An ImageQuant LAS 4000 mini-instrument (GE Healthcare, Chicago, IL, USA) was used to detect protein bands. WB Stripping Solution (Nacalai Tesque) was used to strip membranes of antibodies. The density of protein bands was measured using ImageJ ver. 1.53 (National Institutes of Health, Bethesda, MD, USA). Protein expression was quantified against that of β-tubulin.