A mixture of purified and fragmented bioti nylated cRNA thoroughly and hybridisation controls was hybridised on Affymetrix GeneChip Mouse Genome 430 2. 0 arrays followed by staining and washing in a GeneChip fluidics station 450. To assess the raw probe signal intensities, chips were scanned using a GeneChip scanner 3000. Microar ray data have been deposited in the Gene Expression Omnibus and are accessible through Gene Expression Omnibus accession number GSE33656. Western blot analysis Proteins were isolated from the dissected articular carti lage and subchondral bone pieces using cell extraction buffer supplemented with 1 mM phenylmethanesulfonyl and 5% protease inhibitor cocktail using the Fastprep 24 tissue homogeni ser.

A total of 20 ug of each sample was denatured and separated on a 4 to 12% polyacryla mide Bis Tris gel by electrophoresis using NuPage MES SDS Running buffer. Proteins were transferred to a PVDF membrane. Non specific binding sites were blocked using 5% blottoB in Tris buffered saline with 0. 1% Tween for one hour at room temperature. Inhibitors,Modulators,Libraries Blots were probed overnight at 4 C with the following antibodies, Inhibitors,Modulators,Libraries 1 500 dephospho b catenin sheep antibody, 1 1,000 phospho Smad1 Smad5 Smad8 rabbit antibody, 1 500 anti SFRP1 rabbit antibody, 1 500 mouse DKK2 affinity purified polyclonal goat antibody, 1 1,000 mouse SFRP2 affinity purified polyclonal goat antibody or 1 4,000 anti GAPDH mouse monoclonal Inhibitors,Modulators,Libraries 6C5 in 5% bovine serum albu min in TBS T with 0. 02% sodiumazide. Horseradish per oxidase conjugated donkey anti sheep, mouse anti rabbit, donkey anti goat and goat anti mouse polyclonal antibodies in 5% blottoB in TBS T were used as secondary antibodies.

Blots were visualised using Wes tern Lightning Chemiluminescent Substrate for dephospho b catenin, DKK2, SFRP2, SFRP1 and GAPDH or SuperSignal West Femto Maximum Sensitivity Substrate for phosphorylated Smad. Densitometry analysis was performed with ImageJ Software. Cell culture experiments ATDC5 cells were cultured in maintenance Inhibitors,Modulators,Libraries medium,Hams F 12 mix, 1% antibiotic antimycotic, 5% fetal Inhibitors,Modulators,Libraries bovine serum containing 10 ug ml human trans ferrin and 30 mM sodiumselenite and maintained in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. In FRZB overexpression experiments, ATDC5 cells were transfected with control pcDNA3. 1 or the pcDNA3. 1 full length FRZB construct using lipid based agent Fugene HD.

After 24 hours, selection with 1 mg ml geneticin was initiated. Selection medium was renewed every day for 14 days. Antibiotic resistant U0126 solubility cells were dilution cloned. In Frzb knock down experiments, ATDC5 cells were transfected with control pGIPZ non silencing shRNA mir or with a pGIPZ shRNAmir directed against Frzb using lipo polymeric agent Arrest In. After 24 hours, selection with 0. 5 ug ml puromycin was initiated. Selection medium was renewed every day for seven days. Antibiotic resistant cells were dilution cloned.