Polyclonal rabbit anti mouse igg hrp

Protein was isolated in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phosphatase inhibitor cocktail 3 (p0044, Sigma‐Aldrich), 1x cOmplete protease inhibitor cocktail (11 873 580 001, Roche), and 15 mM sodium orthovanadate (S6508, Sigma‐Aldrich). Protein concentration was determined with the DC protein assay kit. Equal amounts of protein were separated by SDS‐PAGE and proteins were transferred to PVDF membrane. For detection of specific proteins, the following antibodies were used: polyclonal anti‐cathepsin D IgG (1:1000; sc‐10 725, Santa Cruz), monoclonal anti‐Caspase 3 (1:1000; 9664, Cell Signalling) and monoclonal anti‐GAPDH IgG (1:30.000; 10R‐G109A, Fitzgerald). After washing, blots were incubated with polyclonal goat anti‐rabbit IgG‐HRP (1:2000; P0448, Dako), and polyclonal rabbit anti‐mouse IgG‐HRP (1:2000; P0260, Dako). Signals were detected visualized with enhanced chemiluminescence (ECL; NEL120001EA, PerkinElmer) and densitometry has been analysed with ImageQuant LAS 4000 (GE Healthcare). Cathepsin D signals were normalized to respective GAPDH levels.

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).