Anti pt286 cyclin d1

Total proteins were extracted using 1X cell lysing buffer (Cell Signaling Technology). Samples were separated by electrophoresis in SDS- polyacrylamide gels (12%) and transferred to PVDF membranes. Membranes were blocked for 1 h with PBST (PBS plus 0.1% Tween-20) containing 5% non-fat milk. Blots were incubated overnight at 4°C with primary antibodies in PBST containing 5% BSA at the manufacturer's recommended dilution. The antibodies were purchased from the following suppliers: FKBP4 (ProteinTech); anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pT286 Cyclin D1, anti-Cyclin D1, anti-E2F-1, anti-pT37/46 4E-BP1, anti-pS9 GSK-3β, anti-GSK-3β, and anti-mTOR (Cell Signaling Technology); anti-GAPDH and anti-Actin (Santa Cruz Biotechnology); anti-Tubulin and anti-PIK3R2 (Sigma-Aldrich). After washing, blots were incubated with either an anti-rabbit HRP, or an anti-mouse HRP-conjugated antibody (Cell Signaling Technology) for 1 h at 25°C. After washing, blots were developed with Supersignal WEST Pico Plus (Life Technologies SAS).

Total protein extracts (≈ 40 μg) were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences) or Immobilon P membranes (Millipore) that was probed with antibodies whose binding was detected by infrared fluorescence using the LI-COR Odyssey IR Imaging System V3.0 (LI-COR Biosciences) or by chemiluminescence using the Amersham ECL™ Western blotting detection reagent (Amersham Life Sciences) and X-ray Films (AGFA). Primary antibodies used were: mouse monoclonal anti-DYRK1A (1:500; Abnova Corporation or 1:1000; Santa Cruz), anti-p27 (1:500; BD Biosciences), anti-p21 (1:200; Santa Cruz), anti-vinculin (1:5000; Sigma-Aldrich) and anti-Cyclin D1 (1:200, Calbiochem); rabbit polyclonal anti-Cyclin D1 (1:2,000; Thermo Scientifics), anti-actin (1:5000; Sigma-Aldrich), anti-retinoblastoma (1:500; BD Biosciences), anti-HSP90 (1:2000; BD Biosciences), anti-pT286-cyclin D1 (1:500; Cell Signalling), anti-GFP (1:1000, Roche) and anti-RCAN1 (1:1000). Polyclonal HA antibody conjugated to agarose beads was from Santa Cruz. Secondary antibodies for infrared fluorescent detection were goat anti-mouse IgG IRDye-800CW and goat anti-rabbit IgG IRDye-680CW, and for chemiluminescence detection were rabbit anti-mouse and goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2000; Dako).