pCMV6-AN-GFP Mammalian Expression Vector

Snippet: idified atmosphere (∼95%). To stably express TurboGFP (excitation/emission max = 482/502 nm), U87-MG cells were transduced with pCMV6-AN-GFPa shRNA lentiviral vector with an N-terminal tGFP tag (Origene, PS100019), at a concentration of 1 mg/mL in TE buffer, pH 8.0. …

Snippet: The human complementary DNA (cDNA)‐open reading frame (ORF) clone of MDK (RG221818, ORIGENE), the blank vector (PS100019, ORIGENE), and TurboFectin 8.0 (transfection reagent) were purchased from OriGene Technologies (Rockville, MD, USA)

Snippet: Methods. DNA constructs. CYLD, TARDBP and FUS mutations were introduced into the pCMV6-CYLD, pCMV6-TARDBP and pCMV6-FUS constructs (Origene) by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent)

Snippet: entomophaga and PTP-HVR of TccC1 (AKB86704.1) of Yersinia pestis KIM10+ were synthesized by Ruibio BioTech. The coding sequences of GFP and RFP were cloned from pCMV6 vectors (PS100019 and PS100033, Origene). TcdA1 of Photorhabdus luminescens W14 was cloned into RSFDuet-1 (Novagen). All TcB-TcC fusion proteins were constructed incorporating a 5-aa linker region encoding Arg-Gly-Ser-Arg-Pro, and c

Snippet: Iroquois homeobox 1 (IRX1) belongs to the Iroquois homeobox family known to play an important role during embryonic development. Interestingly, however, recent studies have suggested that IRX1 also acts as a tumor suppressor. Here, we use homozygous knockout mutants of zebrafish to demonstrate that the IRX1 gene is a true tumor suppressor gene and mechanism of the tumor suppression is mediated by repressing cell cycle progression. In this study, we found that knockout of zebrafish Irx1 gene induced hyperplasia and tumorigenesis in the multiple organs where the gene was expressed. On the other hands, overexpression of the IRX1 gene in human tumor cell lines showed delayed cell proliferation of the tumor cells. These results suggest that the IRX1 gene is truly involved in tumor suppression. In an attempt to identify the genes regulated by the transcription factor IRX1, we performed microarray assay using the cRNA obtained from the knockout mutants. Our result indicated that the highest fold change of the differential genes fell into the gene category of cell cycle regulation, suggesting that the significant canonical pathway of IRX1 in antitumorigenesis is done by regulating cell cycle. Experiment with cell cycle blockers treated to IRX1 overexpressing tumor cells showed that the IRX1 overexpression actually delayed the cell cycle. Furthermore, Western blot analysis with cyclin antibodies showed that IRX1 overexpression induced decrease of cyclin production in the cancer cells. In conclusion, our in vivo and in vitro studies revealed that IRX1 gene functionally acts as a true tumor suppressor, inhibiting tumor cell growth by regulating cell cycle. …All rights reserved.;;1:1000, Abcam, EPR2241), rabbit anti-cyclin E1 (1:1000, Abcam, EP435E), mouse anti-cyclin B1 (1:1000, Santacruz, GNS1), mouse anti-cyclin A (1:1000, Santacruz, B-8), anti-human IRX1 (1:500, Abcam, ab66310), mouse anti-turboGFP (1:1000, Origene, TA150041