Rabbit anti psmb5

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).

Tet-on inducible RGC5 cell lines were treated with Dox for 24 h to induce moderate expression of wild-type, P370L, or Q368X myocilin-GFP. The total cell lysate was subjected to Western blotting using rabbit anti-PSMB5 (Abcam, Cambridge, MA), rabbit anti-LC3 (MBL, Farmingdale, NY), or rabbit anti-GAPDH. The level of PSMB5 or LC3 normalized to that of GAPDH in induced cells was compared to that in non-induced control cells.
For immunofluorescence, cells induced with Dox were fixed and stained with rabbit anti-PSMB5 or rabbit anti-LC3. Cy5-goat anti-rabbit IgG (Jackson ImmunoResearch, 1∶200) was used as the secondary antibody.