Mouse monoclonal anti actin

Specimens were lysed in a buffer supplemented with a protease inhibitor cocktail (Roche) [14 (link)]. Concentrations of proteins were measured with the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA) using a spectrophotometer. Proteins (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA). The following primary antibodies were incubated overnight with the blocked membranes: rabbit polyclonal anti-COL2A1, rabbit polyclonal anti-MMP-13 (Santa Cruz Biotechnology), goat polyclonal anti-BMAL1, goat polyclonal anti-CLOCK, rabbit polyclonal anti-OPG, goat polyclonal anti-PER1, goat polyclonal anti-PER2, goat polyclonal anti-RANKL, mouse monoclonal anti-actin, rabbit polyclonal anti-GAPDH, and rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with the secondary antibody. Finally, specific bands were visualized with chemiluminescent reagents (Pierce Biotechnology, Rockford, IL, USA) and quantified using the ImageJ software.

Minimum essential medium (MEM), Ham’s F-12 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). Mouse monoclonal anti-actin (catalog number 3700), rabbit polyclonal anti-calpain (catalog number 2539), anti-calpastatin (catalog number 4146) and horseradish peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG antibody were supplied by cell signaling (Beverly, MA, USA). Enhanced chemiluminescence (ECL) plus western blotting reagent was purchased from Amersham Biosciences (Piscataway, NJ, USA). The human dopaminergic neuroblastoma (SH-SY5Y) cell line was obtained from American Type Culture Collection (Manassas, VA, USA). SH-SY5Y cells are a thrice cloned subline of bone marrow biopsy-derived line SK-N-SH. SH-SY5Y cell has dopamine-β-hydroxylase activity and express tyrosine hydroxylase. 8-Hydroxyquinoline (99%), clioquinol (≥95%), and nitroxoline (96%) were purchased from Sigma-Aldrich (St Louis, MO, USA).