in 200 of PBS for 10 minutes at room temperature with rotation. The sates were incubated with washed Dynabead–antibody comp exes for 10 minutes at room Piperine temperature with rotation to a  ow antigen to bind to the comp exes. The supernatants from this reaction were co  ected, concentrated to 50% vo ume by centrifuga  spin fi ter prior to ana ysis as the unbound fraction. The Dynabead–antibody–antigen comp exes were washed 3 times, and the antigens were e uted with 20 of e ution buffer (50 mM g ycine, pH 2.8, a ong with 10 of NuPAGE  DS Samp e Buffer [Invitrogen] and a reducing agent), fo  owed by heating for 10 minutes at 70°C.

E uted supernatants (bound fraction) were co  ected in a magnetic fie d apparatus, and a iquots were  oaded by equiva ent vo ume onto Novex 4–12% gradient SDS-PAGE ge s for vidarabine Western b ot ana ysis. Samp es were processed by e ectrophoreses and e ectrob ot as described above and ana yzed by Western b otting using rabbit anti–MT4-MMP IgG (0.2 g/m ; Santa Cruz Biotechno ogy). The experiment was performed 3 times, with simi ar resu ts obtained in each experiment. Statistica  ana ysis. A   data were obtained from at  east 3 independent experiments, each performed in trip icate. Statistica  significance was determined using Student’s t-test. P va ues  ess than 0.05 were considered significant. Primer sets for rea -time RT-PCR amp ification and buy Naringin quantification of bovine ADAMTS-4, ADAMTS-5, and GAPDH were designed.

Amp ification efficiencies for each primer set were determined a  owing the comparison of mRNA copy numbers between treated and contro  cu tures, which were norma ized to GAPDH. Chondrocytes were incubated for 24 hours under serum-free conditions in the absence of HA o igosaccharides or for 6, 12, 24, or 48 hours in the presence of 250 g/m  of HA o igosaccharides and then processed for tota  RNA. As ear y as 3 hours of Salinomycin  inhibitor incubation with HA o igosaccharides, there was a 2.9-fo d increase in ADAMTS-4 mRNA copy number as compared to untreated contro  chondrocytes (Figure 1A). This effect of HA o igosaccharides was time-dependent, with the maximum enhancement observed at the 24-hour time point (11.3-fo d increase). As shown in Figure 1B, treatment with HA o igosaccharides a so produced a statistica  y significant increase in ADAMTS-5 mRNA expression by 12 hours, reaching a maxima  2.9-fo d increase at 24 hours. HA o igosaccharide–mediated stimu ation of aggrecanase mRNA was transient, and copy numbers had returned to basaeve s at the 48-hour time point (Figures 1A and B).

Hao igosac charides effected a concentrationdependent increase in aggrecanase, reaching statistica  significance at 250 g/m  fo  owing 6 hours of treatment and reaching 100 g/m  fo  owing 24 hours of treatment (Figures 1C and D). As a contro , the same HA o igosaccharides were predigested with chondroitinase ABC to generate HA disaccharides. Incubation of chondrocytes with HA disaccharides at a concentration of 250The maximum  health care industry eve s of stimu ation of ADAMTS-4 and ADAMTS-5 mRNA induced by HA o igosaccharides (11.2-fo d and 2.8-fo d, respective y) (Figure 1) were substantia , but were  ower than the va ues obtained in para  e  cu tures treated with 10 ng/m  of I -1 for 24 hours. In these ce  s, I -1 treatment resu ted in a 22.