Investigating NFAT1 Regulation of MDM2

JapA was obtained as previously reported [13 (link)], with the purity being > 95% as confirmed by IR, ESI-MS, NMR, and HPLC/MSⁿ [16 (link)–17 (link)]. All chemicals and solvents were of the highest analytical grade available. The anti-human NFAT1 (1/NFAT-1) antibody was from BD Biosciences (San Jose, CA). The anti-human COX-2 (C-20) and c-Myc (0.N.222) antibodies were from Santa Cruz Biotechnology Inc. (Dallas, TX). The anti-human MDM2 (Ab-2) antibody was from Calbiochem (Billerica, MA). The anti-human ubiquitin (6C1) and β-actin (AC-15) antibodies were from Sigma (St. Louis, MO). Goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) were obtained from Bio-Rad (Hercules, CA). The human full-length MDM2 P2 promoter reporters were kind gifts from Dr. J.P. Blaydes (Southampton General Hospital, UK). Vectors expressing HA-NFAT1, CA-NFAT1, and DN-NFAT were kindly provided by Dr. C.W. Chow (Yeshiva University). NFAT1 siRNA or control siRNA were from Thermo Scientific (Rockford, IL). Both plasmids and siRNAs were transfected into cells using the same protocols as we reported previously [57 (link)].

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Qin J.J., Wang W., Voruganti S., Wang H., Zhang W.D, & Zhang R. (2015). Inhibiting NFAT1 for breast cancer therapy: New insights into the mechanism of action of MDM2 inhibitor JapA. Oncotarget, 6(32), 33106-33119.

Publication 2015

anti-human NFAT1 (1/NFAT-1) antibody Goat anti-mouse IgG (H+L) goat anti-rabbit IgG (H+L) control siRNA

Actin Anti igg Antibodies Antibody C myc Cells Cox 2 Gifts Goat Hplc Human Human ubiquitin Mdm2 Mouse Plasmids Rabbit Sirnas Solvents Vectors

Corresponding Organization :

Other organizations : Texas Tech University, Texas Tech University Health Sciences Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Jiao Tong University

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Protocol cited in 3 other protocols

Variable analysis

independent variables

NFAT1 siRNA or control siRNA
Plasmids expressing HA-NFAT1, CA-NFAT1, and DN-NFAT

dependent variables

Expression levels of NFAT1, COX-2, c-Myc, and MDM2 proteins

control variables

All chemicals and solvents were of the highest analytical grade available
Anti-human NFAT1 (1/NFAT-1), COX-2 (C-20), c-Myc (0.N.222), MDM2 (Ab-2), ubiquitin (6C1), and β-actin (AC-15) antibodies
Goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) secondary antibodies
Human full-length MDM2 P2 promoter reporters
Transfection protocols as reported previously [57]

positive controls

Not explicitly mentioned

negative controls

Control siRNA

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