Rabbit anti proteasome 20s α β

Immunofluorescence staining of tissue sections and cultured cells were performed as reported before43 (link). Antibodies used in the current study included: rabbit anti-proteasome 20S α + β (Abcam, Cambridge, MA), rabbit anti-PSMB5 (Abcam), mouse anti-nestin (Millipore, Billerica, MA), mouse anti-BrdU (Abtech Biotechnology, Richmond, VA), mouse-anti Tuj1 (Covance, Richmond, CA), Alexa Fluor 555 goat anti-mouse IgG (Invitrogen) and Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen). Images were captured by a fluorescent microscope (Model BX51, Olympus, Japan), and analyzed by Image J software.

Total proteins were extracted using the RIPA lysis buffer supplemented with the protease inhibitor cocktail (Thermo Fischer, Pittsburgh, PA). Protein concentrations were determined by a BCA Protein Assay kit (Beyotime). Equal amounts of proteins were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Blots were incubated with the 5% milk for 1 hr at room temperature, and probed overnight at 4 °C with one of the following primary antibodies: rabbit anti-proteasome 20S α + β (Abcam), rabbit anti-PSMB5 (Abcam), rabbit anti-CDK4 (Abtech Biotechnology), rabbit anti-CyclinD1 (Epitomics, Burlingame, CA) and mouse-anti β-actin (Sigma-Aldrich). On the next day, membranes were thoroughly rinsed in the phosphate-buffer saline (PBS) containing 0.1% Tween-20 (PBST), and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies for 1 hr. Bands were then visualized by the ECL detection kit (GE Healthcare Life Science, Pittsburgh, PA), and documented on films. Intensities of bands were analyzed by densitometry using the QuantityOne software (Bio-Rad, Hercules, CA).