Isotype control antibody

RNP-IP experiments were performed as described previously [14 (link), 27 (link)]. Briefly, UM74B cells grown under normal conditions were UV cross-linked (200mJ/cm²) and lysed with polysome lysis buffer. A minimum of 1mg of total protein was added to Protein A/G (Santa Cruz Biotechnology) beads coated with 1.4μg of CELF1 (EMD Millipore) or isotype control antibody (Santa Cruz Biotechnology). Lysates were incubated with antibody for 2 hours at 4°C and RNA was isolated using Trizol Reagent (Life technologies).

Cells were scraped from culture dishes and centrifuged to pellet cells. Cell pellets were lysed on ice for 1 h in immunoprecipitation (IP) buffer (250 mM NaCl, 50 mM Tris, 0.5 mM EDTA, 0.5% NP-40) with protease/phosphatase inhibitor added. For the anti-VP16 immunoprecipitation, 10 mM N-Ethylmaleimide was added to the IP Buffer immediately before use. Lysates were centrifuged to remove cell debris and the soluble portion was precleared with Isotype control antibody (Santa Cruz) and protein A/G conjugated beads (Santa Cruz) for 1 h with agitation at 4 °C. Beads were pelleted by centrifugation and lysates were moved to new tubes. The lysates were then incubated with isotype or specific antibody (5 µg per 1.7 mL tube with 1 mL lysate) on ice for 1 h. 20 µL of protein A/G beads were added and samples were agitated at 4 °C overnight. The beads containing the immunoprecipitated proteins were pelleted by centrifugation and the unbound portion decanted. The beads were washed four times in IP buffer prior to processing for immunoblot analysis.

Tumor cells were washed with 1×phosphate buffered saline (PBS) buffer containing 20 g/L bovine serum, and then were preincubated with 5% powdered skim milk for 30 min to block nonspecific binding. For surface staining of P-gp and MRP1, aliquots of cells were incubated with fluorescein isothiocyanate (FITC)-anti human P-gp, MRP1 (Abcam, Cambridge, UK) or an isotype control antibody (Santa Cruz Biotech, Santa Cruz, CA) for 40 min at 4°C at the recommended dilutions. For detection of α-2, 6 sialylation, cell lysates were incubated with FITC-Sambucus nigra (SNA) lectin (Vector Laboratories, Inc). After repeated centrifugation at 1000 r/min, labeled cells were resuspended in 0.2 ml PBS and were analyzed with FACSCalibur (BD Biosciences, San Jose, CA, USA). Fluorescence intensity was measured by Cell Quest software.

The mitochondrial membrane potential was measured using a rhodamine (Rho123) based assay, and monitored by flow cytometry. hASC and osteoblast were incubated in a Rho 123 solution (100 mg/mL) diluted in DMSO (5 μg/mL) in a 5% CO₂ incubator for 30 min.
After washing with PBS, the cells were analyzed using a Guava^®easyCyte Flow Cytometer. A total of 10000 cells/sample were analyzed and the mean fluorescence intensity and percentage of cells in each population was recorded.
For the expression of cytochrome-c, caspase 3, P27, cyclin D1 markers, and for the evaluation of autophagy (p62), the cells were first fixed with 1% paraformaldehyde and then permeabilized with 0.02% Triton X-100, then stained with anti-cytochrome c, anti caspase 3, anti P27, anti-and cyclin D1 and anti p62 antibodies, or isotype control antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The percentages of cells expressing the respective markers were quantified by flow cytometry (Guava^®easyCyte Flow). A total of 10,000 cells per sample was analyzed and the mean fluorescence intensity and percentage of cells in each population were determined.

Rat joint tissue was fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier and embedded in paraffin. The sections (7 μm) were stained with hematoxylin and eosin (H&E) and toluidine blue to detect proteoglycans. For immunohistochemistry, rat joint sections were blocked with 1% normal goat serum and stained with antibodies to IL-6 (1 μg/mL, Santa Cruz, CA, USA), COX-II (200 ng/mL, Santa Cruz, CA, USA) and isotype control antibody (1 μg/mL, Santa Cruz, CA, USA) at 4 °C overnight. After three washes in PBS, the secondary antibody (biotin-labeled goat anti-rabbit IgG) was applied for 1 h at room temperature. Staining was detected with 3, 3′-diaminobenzidine tetrahydrochloride and the sections were then counterstained with H&E and observed under a light microscope.