OBs were treated with MSU or ve hicle at day 8. At day 20, cells were fixed for 20 minutes with buffered formalin and then stained for always find useful information 20 minutes with 40 mM ARS, pH 4. 0 to 4. 2 at room temperature. After four washes with distilled H2O, ARS was extracted, as previously described. In brief, ARS cells were incubated 30 minutes with acetic acid and then heated 10 minutes Inhibitors,Modulators,Libraries at 85 C. pH was re stored at 4. 2 with NaOH, and ARS absorbance was read at 405 nm. MMP activity Evaluation of generic matrix metalloproteinases was assessed with the SensoLyte Generic MMP assay kit that detects the activity of a variety of MMPs, including MMP 1, 2, 3, 7, 8, 9, 12, 13, and 14. Five FAM and QXL520, labeled FRET peptide substrates, Inhibitors,Modulators,Libraries were used for continuous measurement of the enzyme activities.
On the cleavage of the FRET peptide by MMPs, the fluorescence of 5 FAM was recovered and monitored at excitation emission wavelengths of 490 nm 520 nm. Confluent Inhibitors,Modulators,Libraries OBs were treated 24 hours with or without 0. 5 mg MSU in MEM containing 1% FBS. Medium was then centrifuged 2 minutes at 10,000 rpm, and 50 ul of supernatant was added to 50 ul of MMP substrate for 20 minutes. MMP activity in MSU stimulated cells was compared with MMP activity in untreated cells. RNA isolation and real time PCR OB total RNA was isolated by using Trizol. In brief, around 106 confluent cells, stimulated with MSU or vehicle, were washed in PBS and then homogenized in 1 ml Trizol. Total RNA was then extracted, according to the manufacturers protocol. Reverse transcription and real time PCR were performed essentially as previously described in.
In brief, first strand cDNA synthesis was performed by using 1 ug of total RNA with Superscript II in recommended conditions, with 10 Inhibitors,Modulators,Libraries ng of random hex amers. Amplification of osteoblast cDNA was carried out in a Rotor Gene 3000 operated with Rotor Gene software version 6. 0. 19. Each sample consisted of 50 ng cDNA, 1. 3 mM MgCl2, 0. 2 mM dNTP, 500 nM primers, 0. 5 unit of Taq polymerase, and Sybr Green dye in a reaction volume of 20 ul. Amplification conditions were as follows 95 C, 60 C, 72 C, 35 cycles. Specificity of each reaction was ascertained by performing the Melt procedure after completion of the amplification protocol, according to the manufacturers instructions. Proteome profiler assay Signaling pathways were investigated by using the Proteome Profiler arrays.
The Human Phospho Inhibitors,Modulators,Libraries Kinase array is a nitrocellulose membrane where antibodies against 46 kinase phosphor ylation sites have been spotted in duplicate. Cell lysates from untreated, 5 minute, 20 minute, and 1 hour MSU selleckchem activated cells were prepared in lysis buffer provided with the proteome profiler. In total, 250 ug of protein was used for each array and incubated with the nitrocellu lose membrane array overnight at 4 C.