Anti-FLAG tag antibody, clone O4 [DyLight® 488]

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.

A human ciliopathy gene codes for Polycystin-2 (Pkd2), a non-selective cation channel. Here, the Pkd2 channel was explored in the ciliate Paramecium tetraurelia using combinations of RNA interference, over-expression, and epitope-tagging, in a search for function and novel interacting partners. Upon depletion of Pkd2, cells exhibited a phenotype similar to eccentric (XntA1), a Paramecium mutant lacking the inward Ca2+-dependent Mg2+ conductance. Further investigation showed both Pkd2 and XntA localize to the cilia and cell membrane, but do not require one another for trafficking. The XntA-myc protein co-immunoprecipitates Pkd2-FLAG, but not vice versa, suggesting two populations of Pkd2-FLAG, one of which interacts with XntA. Electrophysiology data showed that depletion and over-expression of Pkd2 led to smaller and larger depolarizations in Mg2+ solutions, respectively. Over-expression of Pkd2-FLAG in the XntA1 mutant caused slower swimming, supporting an increase in Mg2+ permeability, in agreement with the electrophysiology data. We propose that Pkd2 in P. tetraurelia collaborates with XntA for Mg2+-induced behavior. Our data suggest Pkd2 is sufficient and necessary for Mg2+ conductance and membrane permeability to Mg2+, and that Pkd2 is potentially a Mg2+-permeable channel.