The 3.2kb mouse calbindin-D28k promoter reporter, kind gifts from Dr. Harry T. Orr (University of Minnesota) [25 (link)] as well as the promoter deletion constructs were cloned in the pGL3 basic vector (Promega). Bioinformatics analysis of murine 3.2kb calbindin-D28k promoter was performed using the public domain Transcription Element Search System (TESS) software (http://www.cbil.upenn.edu/cgi-bin/tess/tess?RQ=WELCOME ). NFAT expression vectors were kindly provided by Chi-Wing Chow (Albert Einstein College of Medicine). All transient transfection experiments were done in 96 well plates using Fugen-6 (Roche) or lipofectamine 2000 (Invitrogen) following manufacturer's recommendation. The pSV-β-galactosidase expression vector (Invitrogen) was a normalization control. Luciferase activity was measured 24 hours post transfection, using the manufacturer's protocol in the FLUOstar Omega luminometer (BMG LABTECH).
Fugen 6
Manufactured by Roche
Sourced in United States
The Fugen 6 is a laboratory instrument designed for the analysis of DNA and RNA samples. It is capable of performing real-time PCR (polymerase chain reaction) experiments. The Fugen 6 provides accurate and reliable results for a variety of applications in the life sciences and diagnostic fields.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fluostar omega luminometer, by BMG Labtech (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti gpr14, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti vegf receptor 2 antibody, by Abcam (1 mentions)
Pe rat anti mouse cd31, by BD (1 mentions)
Anti hif 1α antibody, by Proteintech (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Trizol, by Takara Bio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
5 protocols using fugen 6
1
Mouse Calbindin-D28k Promoter Assay
2
Evaluating HAND1 Overexpression Effects
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Cells were plated at 3.0 × 105 per well using six-well plates, and transfected with either pcDNA4-HAND1 or empty vector (2.0 μg) using Fugen 6 (Roche, Indianapolis, IN, USA). The cells were replated in triplicates and cultured for 10–15 days in complete DMEM medium containing Zeocin (100 μg/ml) and stained with Gentian Violet after methanol fixation. For anchorage-independent growth, cells were transfected as above, and suspended in DMEM containing 0.3% agar, 10% fetal bovine serum, and layered on DMEM containing 0.6% agar, 10% FBS and Zeocin (100 μg/ml) in six-well plate at 48 h post-transfection. Colony formation was assessed at 4 weeks post-transfection.
3
Cell Culture of Tumor Lines
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Tumor cell lines were grown in appropriate medium with 10% fetal bovine serum (FBS) 2 mM glutamine, 50 mg/mL penicillin, 50 mg/mL streptomycin (complete medium) at 37 °C in 5%CO2. PES43 human melanoma cells derived from a lung metastases of melanoma patient were grown in Iscove’s Modified Dulbecco’s Medium (IMDM). FB-1 human anaplastic thyroid cancer cell line (kindly provided by Dr. Melillo, University of Naples, Federico II, Italy) were grown in Dulbecco’s Modified Eagle’s Medium (DMED). B16-CXCR4 murine melanoma cells were obtained by transfection of B16 with pYF1-fusin plasmid containing human CXCR4 gene (kindly provided by Dr Aloj, NCI “Pascale”, Naples, Italy) using Fugen 6 (Roche Applied Science, Indianapolis, IN) in according to manufacturer’s instruction and grown in complete medium supplemented with 100 µg/mL G418. CHO and CHO cells transfected with human CXCR4 (CHO-CXCR4) were kindly provided by Dr. David McDermott (NIH, Bethesda, USA) and grown in complete medium supplemented with 1 mg/mL G418.
4
LRP6 Overexpression in AD293 Cells
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AD293 cells were stably transfected with human UTR/GPR14 pcDNA3.1+ (Missouri S&T cDNA Resource Center, Rolla, MO, USA) using FUGEN6 (Roche, Diagnostics Operations Inc., Indianapolis, IN, USA). Positive cells were selected by G418 (Sigma-Aldrich). UTR overexpression was identified by western blot with anti-GPR14 (Santa Cruz, Dallas, TX, USA). Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion. For western blot analysis, isolated proteins were size-fractionated by SDS–PAGE and transferred to polyvinylidene difluoride membrane. And then the membranes were incubated with primary antibodies followed by incubation with a Horseradish Peroxidase (HRP)-conjugated secondary antibody (Jackson, West Grove, PA, USA). All antibodies used here were: phosphor-JNK, JNK and phosphor-LRP6 (1490), LRP6 (Cell Signalling, Danvers, MA, USA), GAPDH (Kangchen, Shanghai, China).
5
Endothelial Cell Differentiation and Angiogenesis
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Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD Pharmingen™); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).
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