Anti-DYKDDDDK polyclonal antibody

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.

To comprehensively analyze bacterial gene function, it is important to simultaneously generate multiple genetic modifications within the target gene. However, current genetic engineering approaches, which mainly use suicide vector- or lambda red homologous recombination-based systems, are tedious and technically difficult to perform. Here, we developed a flexible and easy method to simultaneously construct multiple modifications at the same locus on theSalmonella entericaserovar Typhimurium chromosome. The method combines an efficient seamless assembly systemin vitro, red homologous recombinationin vivo, and counterselection markersacB. To test this method, with the seamless assembly system, various modification fragments for target genescpxR,cpxA, andacrBwere rapidly and efficiently constructedin vitro.sacBKancassettes generated via polymerase chain reaction were inserted into the target loci in the genome ofSalmonellaTyphimurium strain CVCC541. The resulting pKD46-containing kanamycin-resistant recombinants were selected and used as intermediate strains. Multiple target gene modifications were then carried out simultaneously via allelic exchange using various homologous recombinogenic DNA fragments to replace thesacBKancassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 x FLAG tagging of the target genes. This method can be used in any bacterial species that supportssacBgene activity and lambda red-mediated recombination, allowing in-depth functional analysis of bacterial genes.