Anti ago2 antibody

Ribosomal protein S3- and calnexin-specific antibodies were from Cell Signaling Technology; anti-Ago2 antibody was from Abnova; anti-HA antibody was from Roche Applied Science; GAPDH- and β-actin-specific antibodies were from Sigma. RL-con, RL-3×bulge-let7a, and RL-3×bulge-miR-122 plasmids were kind gifts from Prof. Witold Filipowicz. Fragments encoding HMGA2 3′-UTR/HMGA2 3′-UTR mut were cloned in the XbaI and NotI sites of RL-con plasmids to get RL-HMGA2 3′-UTR/RL-HMGA2 3′-UTR mut plasmids. Tet-on advanced plasmids were from Clontech. The fragment encoding RL-3×bulge-miR-122 was re-cloned in the NheI and NotI sites of the pTRE-tight-BI vector from Clontech to get the iRL-3×bulge-miR-122 plasmid. For experiments using tetracycline-inducible constructs, induction was done using 300 ng/ml doxycycline (Sigma). Treatment with thapsigargin (Sigma) was done overnight. Expression of the HA- and N-HA-tagged versions of Ago proteins was achieved by transfecting cells with the desired plasmids obtained either from Prof. Witold Filipowicz or from Prof. Gunter Meister. SiRNA against the 3′-UTR of Ago2 was obtained from Eurogentec.

HUVEC were lysed and then immunoprecipitated with anti-Ago2 antibody (Abnova, Taiwan, China) by protein G Sepharose beads as previously described [36 (link)].