Molecular analysis of the articular cartilage - subchondral bone to corroborate the microarray data. (A) Real-Time PCR analysis of tibia articular cartilage and subchondral bone from frizzled-related protein-knockout (Frzb-/-) mice compared to wild-types. Frzb was virtually absent and secreted frizzled-related protein 1 (Sfrp1) and secreted frizzled-related protein 2 (Sfrp2) were significantly upregulated in Frzb-/- samples compared to wild-type samples. There was a trend of up-regulation for dickkopf homolog 2 (Dkk2) (one outlier). Data are shown as relative expression values versus hypoxanthine guanine phosphoribosyl transferase (Hprt) (2-ΔCt) (n = four Frzb-/- and five wild-type samples; All experiments were performed in duplicate; Mann-Whitney test: P = 0.016 for Frzb, P = 0.032 for Sfrp1, P = 0.016 for Sfrp2 and P = 0.2 for Dkk2). (B-C) Western blot and densitometry analysis of proteins extracted from tibia articular cartilage and subchondral bone showed no consistent change in active (dephospho) β-catenin (80 kDa) (B) and phosphorylated (phospho) Smad 1/5/8 (mothers against decapentaplegic homolog) (55 kDa) (C) between three wild-type (lane 1-3) and in three Frzb-/- (lane 4-6) samples. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (37 kDa) Western blot is shown as loading control. Quantitative analysis was performed with Image J software. Data are shown as the ratio of the mean optical density (OD) for β-catenin or P-Smad/the mean OD of GAPDH (n = three samples/group; Mann-Whitney test: P > 0.05 for β-catenin and for P-Smad).