Cyclin a2 antibody

UA was bought from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin solution (EDTA) were bought from Gibco (Invitrogen, Grand island, NY). COX-2 (catalog no. 160106) and mPGES-1 (catalog no. 160140) antibodies were bought from Cayman Chemicals (Ann Arbor, MI). Cyclin A2 antibody (catalog no. ab7956) was purchased from Abcam (Cambridge, MA). Cyclin D1 antibody (catalog no. 2978) and GAPDH antibody (catalog no. ab9485) were purchased from Cell Signaling Technology (Danvers, MA). The PGE₂ enzyme immunoassay kit (catalog no. 514010-96) was provided by Cayman Chemicals (Ann Arbor, MI). COX-2 inhibitor NS-398 (catalog no. s1772) was from Beyotime (Shanghai, China).

Immuno-precipitation using anti-cyclin A2 antibody was performed at 48 h or 72 h after treatment. Cells were lysated by the lysis buffer containing 150 mM KCl, 25 mM Tris-HCl, pH7.4, 5 mM EDTA, 0.5% Triton X-100, 5 mM dithiothreitol (DTT), PMSF and cocktail. The supernatant was mixed with cyclin A2 antibody (Abcam) overnight at 4 °C, and then co-cultured with beads (santa cruz) for 2–4 h at RT. The beads were washed five times in lysis buffer followed by western blotting (WB) analysis.