Anti phosphorylated rb

Western blots were performed as described [27 (link)]. Briefly, cells were lysed in SDS sample buffer without dithiothreitol and bromophenol blue and then cleared of debris by centrifugation. Protein concentration in the lysates was determined by a bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL). Dithiothreitol was added to a final concentration of 125 mM and samples were boiled for 10 minutes. Equal amounts of protein were subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes, and the membranes were incubated overnight with one of the following primary antibodies: anti-Sirt6 (1 : 1000), anti-eNOS (1 : 1000, BD Biosciences, San Jose, CA), anti-tubulin (1 : 10000, Sigma-Aldrich, St. Louis, MO), anti-p21 (1 : 200, Santa Cruz Biotechnology, Dallas, TX), and anti-phosphorylated Rb (1 : 1000, Cell signaling) followed by appropriate second antibodies for 1 hour. Immunoreactive proteins were detected using the enhanced chemiluminescence (ECL) system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).

Total proteins from BMOL cells cultured for 20 or 40 days in William's medium with 5% FCS with or without IL-17 were extracted with ice-cold RIPA lysis buffer and quantified with the BCA kit (Pierce). Proteins were electrophoresed on SDS-PAGE gels and transferred to a nitrocellulose membrane (Invitrogen) after blocking with 5% BSA. Ten µg of proteins were added to each well. Protein detection was done using the following antibodies: anti-cyclin D, anti-cyclin E, anti-p21 from Santa Cruz Biotechnologies, anti-Retinoblastoma (RB), anti-phosphorylated RB and anti-c-Raf from Cell Signaling Technology and anti-β-actin from Sigma-Aldrich. The membrane was incubated with the secondary antibodies (Horseradish peroxidase) and signals were detected with ECL reagent (GE Healthcare).  The band intensity was measured by using Image J software (NIH).

Western blotting analysis was performed with standard techniques, as described previously [3 (link)]. Cell proteins were extracted by a modified RIPA buffer containing 0.5% sodium dodecyl sulfate (SDS) in the presence of a proteinase inhibitor cocktail (Roche, IN, USA). Polyacrylamide gel electrophoresis (PAGE) was performed to separate cell lysate proteins and then fractionated proteins were transferred onto a PVDF membrane (Amersham Biosciences, NJ, USA). Immonodetection was performed using antibodies including rabbit anti-FOXM1 polyclonal antibody, anti-cyclinD1, anti-p21, anti-LEF1, anti-c-myc, anti-Rb, anti- phosphorylated –Rb, and β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA) at the dilution ratio of 1:1000. The membrane was then incubated with HRP labeled goat anti-rabbit secondary antibody (BosterBio, CA, USA) at the dilution ratio of 1:6000. Anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) served as an internal control. Signals were detected by exposure to films with SuperSignal West Pico Chemoluminescent substrate (Thermo Fisher Scientific, MA, USA).