Odyssey ir imaging system v3

Mice were euthanized by decapitation and their hippocampi were dissected and stored at −80° C. Six hippocampi from TS and CO mice were lysed and homogenized as previously described [68 (link)]. The homogenates were boiled for 10 min and sonicated for 5 cycles of 30 s On/Off at 4° C using a Bioruptor Plus (Diadode) and left on ice for 20 min. The total protein content of each sample was determined following the protocol described by [88 (link)]. Identical amounts of protein from each sample were loaded on a 15% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were incubated with a polyclonal rabbit anti-acetyl histone H4 (06-598, Upstate), polyclonal rabbit anti-tri-methylated histone H4 at K20 (07-463, Upstate) and polyclonal rabbit anti-nucleolin (ab22758, Abcam) overnight at 4° C. To ensure equal loading, the blots were reproved using a mouse monoclonal anti-GAPDH antibody (6C5) (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After extensive washing, immunoblots were developed with goat anti-rabbit IRDye 680RD antibody or goat anti-mouse IRDye800CW (1:10,000; LI-COR Biotechnology, Lincoln, NE, USA)
Protein bands were detected using a LI-COR ODYSSEY IR Imaging system V3.0 (LI-COR Biotechnology) and quantified following the protocol described by [68 (link)].

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).