Insoluble material was removed GDC941 by centrifugation at 15 000 g for 15 min at 4°C. The supernatant was saved and the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). An identical amount of protein (50 μg) for each lysate was subjected to 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. Western blot analysis using phosphospecific anti-JAKs and STATs antibodies was performed with an ECL Western blotting

kit (Amersham, Little Chalfont, UK). Total RNA was extracted from fibroblast-like synoviocytes (FLS) using the RNeasy total RNA isolation protocol (Qiagen, Crawley, UK). Total cellular RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. First-strand cDNA was synthesized from 1 μg of total cellular RNA using an RNA PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. Thereafter, cDNA was amplified using

specific primers for acute phase-SAA (SAA1 + SAA2), respectively. The specific primers used were as follows: A-SAA: forward primer 5′-CGAAGCTTCTTTTCGTTCCTT-3′, reverse primer 5′-CAGGCCAGCAGGTCGGAAGTG-3′; β-actin; and forward primer 5′-GTGGGGCGCCCCAGGCACCA-3′, reverse primer 5′-CTCCTTAATGTCACGCACGATTTC-3′. The product sizes were 300 base pairs (bp) for A-SAA and 234 bp for β-actin. The thermocycling conditions (35 cycles) for the targets click here were as 94°C for 60 s and 53°C for 60 s, and 72°C for 60 s. The PCR products were electrophoresed check details on 2% agarose gels and visualized by ethidium bromide staining. The amplification of the MCP-1 transcripts was performed on a Light Cycler (Roche Diagnostics, Mannheim, Germany) using specific primers. The housekeeping gene fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for verification of equal loading. To study the role of the JAK-3 pathway in rheumatoid

synovitis, we examined JAK-3 phosphorylation levels using immunohistochemical staining of synovial tissues isolated from RA and OA patients. Fig. 1a shows a representative section of synovial tissues from seven independent patients with RA and two with OA. Brown phospho-JAK-3 staining was observed in the rheumatoid synovium, indicating that infiltrating mononuclear cells in the synovial sublining area expressed high levels of phospho-JAK-3. In contrast, few infiltrating cells in the OA synovium expressed phospho-JAK-3. In immunohistochemical analysis using the serial sections, the immunophenotype of the infiltrates expressing phospho-JAK-3 was found to be predominantly CD3+ T cells, however, some of which expressed vimentin partiality in sublining infiltrating cells (Fig. 1b).