Jak stat phospho antibody array

The JAK-STAT-protein profiling was performed using the Jak/Stat Phospho Antibody Array (PJS042) and the antibody Array Assay Kit (KAS02), designed and manufactured by Full Moon Biosystems (Sunnyvale, CA, USA). The proteins microarray analysis was carried out according to the manufacturer's instructions. Briefly, 5X10⁶ BMK-16/myc cells were cultivated on cell culture flasks and were treated with 100 ng/ml of ovine IFN-τ during 15 min; a non-treated cell culture was used as control. After the cells were harvested using a scraper and lysed (with Lysis beads and buffer), the clarified-protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, Ill, USA). One hundred micrograms of cell lysate was labeled with biotin in DMF (10 μg/μl) (N,N-Dimethylformamide), and biotin-labeled were diluted 1:20 in a coupling solution before applying them to the array for conjugations. The antibody microarray was blocked before the incubation with the biotin-labeled cell lysates at room temperature (RT) during 4 hrs. Then, the slides were washed; subsequently, 60 μ of Cy3-streptavidin in detection buffer (0.5mg/ml) were added, and the mix was incubated for 20 min at RT. After the slides were washed with Milli-Q grade water, they were dried by centrifugation before the fluorescence detection.

The following fluorophore-conjugated antibodies were purchased from BD Biosciences (Franklin Lakes, NJ): anti-CD4 (RM4-5), anti-ICOS-1 (D10.G4.1), anti-CXCR5 (2G8). All isotype controls were purchased from BD Biosciences. Anti-Fcγ-R (2.4G2) was produced from a hybridoma. IBA-1(019-19741) was purchased from WAKO (Richmond, VA). Anti-MAP2 (MAB8304) and anti-CXCL13 (AF470) were purchased from R and D systems (Minneapolis, MN). Anti-GFAP (AB5541), Anti-NeuN (MAB377B) and Anti-IL-21 (06-1074) was purchased from EDM Millipore (Hayward, CA). Anti-IL-21R (PA5-19982) was purchased from Invitrogen (Carlsbad, CA). JAK/STAT Phospho Antibody Array was purchased from Full Moon BioSystems (Sunnyvale, CA).