TA strategy for rapid and efficient site-directed mutagenesis.

A simple, rapid, and efficient site-directed mutagenesis method using TA strategy with synthetic mutagenic oligonucleotides is described. Briefly, a 3' A-overhang vector was prepared by polymerase chain reaction (PCR) using a classical Taq polymerase with terminal transferase activity, a reverse vector primer starting the complement nucleotide prior to the 5' end adenosine of the target, and a forward vector primer starting the nucleotide posterior to the 3' end thymidine. The 3' T-overhang mutagenic double-strand oligonucleotide was synthesized and cloned directly into the PCR-amplified 3' A-overhang vector.

Reactive oxygen generated by NADPH oxidase 1 (Nox1) contributes to cell invasion by regulating matrix metalloprotease-9 production and cell migration.

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells.

A reducing and denaturing step maximizes the immunoprecipitations of m-calpain and I-2(PP2A)/SET: an approach toward antibodies that do not work well in immunoprecipitation.

Immunoprecipitation is an elegant method to isolate a specific protein of interest from a complex protein mixture such as cell lysate. We tried to increase the efficiency of m-calpain immunoprecipitation with anti-m-calpain antibodies directed toward denatured antigens that only work for immunoblotting and immunohistochemistry. We found that a reducing and denaturing step prior to immunoprecipitation greatly potentiates the efficiency of the immunoreaction.

The oncoprotein I-2PP2A/SET negatively regulates the MEK/ERK pathway and cell proliferation.

I-2PP2A/SET, the translocation breakpoint-encoded protein expressed in acute undifferentiated leukemia, was identified as an inhibitor of protein phosphatase 2A (PP2A). Induction of exogenous I-2PP2A/SET at a ratio of 1:1 to the endogenous protein resulted in suppression of cell proliferation. In contrast, siRNA-mediated depletion of I-2PP2A/SET resulted in enhanced cell proliferation.

Expression of LUN gene that encodes a novel RING finger protein is correlated with development and progression of non-small cell lung cancer.

LUN is a novel RING finger protein that is highly expressed in the lung and might be a transcriptional regulator of E-cadherin [J. Biol. Chem.

Regulation of amphiphysin1 by mitogen-activated protein kinase: its significance in nerve growth factor receptor-mediated endocytosis.

Amphiphysin1, which can simultaneously bind to dynamin1 and the clathrin adaptor AP-2, is essential for dynamin1 recruitment during receptor-mediated endocytosis, but little is known about its regulatory mechanism. Here, we purified a 120-kDa mitogen-activated protein kinase (MAPK) substrate protein from porcine brains and identified the protein as amphiphysin1. Serine phosphorylation of amphiphysin1 was rapidly induced by nerve growth factor (NGF) in PC12 cells, and the induction was blocked by a MAPK inhibitor.