Immobilon ecl western blotting detection kit reagent

Protein extracts were prepared using RIPA buffer supplemented with 1% protease inhibitor cocktail (Thermo Fisher Scientific Inc.), 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc.) and quantified by BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad Laboratories). After, membranes were blocked for 1h with 5% skim milk in 0.1% Tween PBS and incubated with the specific primary antibodies overnight at 4°C. Then, membranes were treated with the appropriate secondary antibody for 1 h at room temperature. All blots were treated with Immobilon ECL Western Blotting Detection Kit Reagent (EMD Millipore) and developed after exposure to an autoradiography film (VWR International). The following antibodies were used: anti-cyclin E (HE12, Santa Cruz) and anti-actin (691001, Millipore).

Cell extracts were obtained from fresh plates 24 h after incubation with the corresponding growth medium. Then, cells were incubated for 30 min on ice with lysis buffer, scraped, sonicated and centrifuged at 15,000 g for 20 minutes at 4 °C. Supernatants were recovered and the protein content was quantified by the BCA kit (Pierce Biotechnology). Western blot analysis was carried out size-separating an equal amount of protein by electrophoresis on SDS polyacrylamide gels, and then the proteins were electroblotted onto polyvinylidene fluoride transfer membranes (PVDF) (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% of non-fat dry milk in PBS with 0.1% Tween, and then incubated with specific primary antibodies overnight at 4 °C. Next, membranes were treated with the appropriate secondary antibody for 1 hour at room temperature. All blots were visualized on Fujifilm X-ray (VWR International, Radnor, PA, USA) with chemiluminescence detection using Immobilon ECL Western Blotting Detection Kit Reagent (EMD Millipore, Billerica, MA, USA). The antibodies used were CAD (Santacruz Biotechnology), CAD-P (Cell Signaling), PDH (Merck Millipore) PDH-P (Cell signaling) and β-actin (MP Biomedicals). Also, anti-mouse (Dako) and, Anti-rabbit (Amersham Biosciences) secondary antibodies were used.