Las 3000 documentation system

SDS PAGE electrophoresis and blotting were performed as previously described [15] (link). In short, whole tissue or cell lysates were produced in RIPA buffer supplemented with PhosSTOP (Roche) and Protease inhibitor cocktail (Roche). Subsequently samples were boiled in 4× Leammli buffer, including 2% β-mercaptoethanol, for 5 min at 95 °C. SDS-PAGE and Western blotting were performed using the Mini-PROTEAN 3 system (Bio-Rad). Blotted membranes were blocked in 5% BSA/TBS-Tween. Primary antibody labeling was performed overnight at 4 °C. Secondary IgG–horseradish peroxidase (HRP)-conjugated antibodies were applied for 2 h at room temperature. Following incubation with an antibody, blots were washed for 3 × 10 min in TBS-Tween. Images were generated using Supersignal West Dura Extended Duration ECL Substrate (Pierce) and the LAS-3000 documentation system (FujiFilm Life Science). Stripping was performed with Restore Western blot stripping buffer (Pierce). Outputs were normalized for loading and results are expressed as an n-fold increase over the values of the control group in densitometric arbitrary units. Primary antibodies that were used included rabbit polyclonal anti-Dyrk1A (Santa Cruz, 1:500), mouse monoclonal anti-α-tubulin (Sigma, 1:1000). Secondary antibodies included polyclonal rabbit anti-mouse IgG–HRP (DAKO, 1:5000) and polyclonal swine anti-rabbit IgG–HRP (DAKO, 1:5000).