α cyclin d1

Anti (α)-Akt, α-pAkt, α-Axin, α-β-catenin, α-C3, α-c-Myc, α-cyclin D1, α-GSK3, α-GSK3β, α-pGSK3, α-IL17, α-IL17RC, α-IRS1, α-PI3K, α-pPI3K, α-TAU, α-VEGF, α-Wnt-3a, and α-Wnt-10b were purchased from Santa Cruz. α-c/EBPα, α-pc/EBPα, α-ERK1/2, α-pERK1/2, and α-pIRS1 were bought from Cell Signaling Technology. α-GSK3β was obtained from BD Transduction Lab. GAPDH was purchased from ImmunoChemical. IL17A, IL17F and sFRP2 were bought from R and D. VEGF was obtained from Sigma-Aldrich. IL17A or IL17F (500 ng/mL), sFRP2 (250 ng/mL) or VEGF (10 ng/mL) was applied to cell culture. An IL17RC expression vector was purchased from OriGene.

Confluent dedifferentiated chondrocytes were supplemented with 5% v-PL either in the presence or in the absence of bovine serum. At different times from the addition of the supplement, cells were washed with PBS and collected for western blot analysis. Electrophoresis was performed in reducing conditions using 25 μg of protein loaded on a 4–12% NuPAGE Bis-Tris gel (Invitrogen, Italy) as described (Ulivi et al., 2006 (link)). Primary antibodies tested were α-Cyclin D1 (1:250) and α-Actin (1:200) (Santa Cruz Biotechnology, USA), α-phospho Erk1/2 (1:1,000), α-phospho Akt (1:1,000) (Cell Signaling Technology, USA). Secondary HRP conjugated anti-mouse and anti-rabbit antibodies were both diluted 1:5,000 (GE Healthcare, UK), peroxidase conjugated anti goat antibody was diluted 1:10,000 (Jackson Immunoresearch, USA). ECL was purchased from GE Healthcare, UK.
Images were scanned using the Epson perfection 1260 scanner (Epson, Italy) and band densities were quantified using the ImageJ software (http://rsbweb.nih.gov/ij/download.html). Western blots were performed on three different independent primary cultures.

Iscove medium (Iscove MEM), L-glutamine, trypsin/ethylenediaminetetraacetic acid (EDTA), and penicillin/streptomycin were obtained from EuroClone S.p.A. (Milan, Italy). Fetal Calf Serum (FCS), collagenase I, and collagenase II were purchased from ThermoFisher Scientific (Waltham, MA, USA). Ascorbic acid, dexamethasone, β-glycerophosphate, tyazolyl blue (MTT), Alizarin Red S was purchased from Sigma Aldrich (St. Louis, MO, USA). Dispase II was purchased from Roche (Basel, Switzerland).
Antibodies α-Cyclin D1 and α-Actin were from Santa Cruz Biotechnology (Dallas, TX, USA), HIF-1α from BD Bioscience (San Jose, CA, USA), phospho-STAT3 and STAT3 from Cell Signaling Technology (Danvers, MA, USA), horseradish peroxidase (HRP) conjugated anti mouse and anti-rabbit antibodies and enhanced chemiluminescence (ECL) were purchased from GE Healthcare (Chicago, IL, USA), VEGF polyclonal antibody from ThermoFisher Scientific (Waltham, MA, USA). Peroxidase conjugated anti goat antibody (1:10,000) from Jackson Immunoresearch (Cambridgeshire, United Kingdom), TransAM HIF-1 kit was from Active Motif (Carlsbad, San Diego, CA USA), Human VEGF Quantikine ELISA kit was provided by R&D System (Abingdon, UK).