Anti-Flag tag monoclonal antibody [Cy5.5]

Recombinant viruses of strain Ingelvaco PRRS porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus vaccine were produced with two individual small in-frame deletions in nonstructural protein 2 (nsp2; Delta 23 and Delta 87) and also the same deletions supplanted with foreign tags (Delta 23-V5, Delta 23-FLAG, Delta 23-S, Delta 87-V5, Delta 87-FLAG, Delta 87-S). The viruses, but one (Delta 87-FLAG), were stable for 10 passages and showed minimal effects on in vitro growth. Northern hybridization showed that the Delta 23-tagged probe detected intracellular viral genome RNA as well as shorter RNAs that may represent heteroclite species, while the Delta 87-tagged probe detected predominantly only genome length RNAs. When the tagged viruses were used to probe nsp2 protein in infected cells, perinuclear localization similar to native nsp2 was seen. Dual infection of Delta 23-S and Delta 87-S viruses allowed some discrimination of individual tagged nsp2 protein, facilitating future research. The mutants could potentially also be used to differentiate infected from vaccinated animals.

Hepatocellular carcinoma (HCC) is frequently complicated by the occurrence of intrahepatic and extrahepatic metastases, leading to poor prognosis. To improve the prognosis for HCC patients, there is an urgent need to understand the molecular mechanisms of metastasis in HCC. Since protein Serine/Threonine phosphorylation emerges to be an important posttranslational modification critical in signaling process associated with cell proliferation, survival and metastasis, we employed a pair of primary tumor-derived and corresponding lung-metastatic counterparts (PLC/PRF/5-PT and PLC/PRF/5-LM) and aimed to identify these changes using CelluSpot(TM) Serine/Threonine kinase peptide array. Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr(234/237)) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr(234/237) was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr(234/237) expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr(234/237) is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wildtype NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr(234/237) phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway. Identification of CDK1/p-NPM/ROCK2 signaling pathway provides a novel target for molecular therapy against HCC metastasis.