Antibody-based Immune Cell Analysis

Antibodies used in this study are listed in Supplementary Table 4, and additional antibodies for cell stimulation, immunoblot and flow cytometry were as described^{39 (link)}. APC-Annexin V, FITC-Annexin V and Propidium Iodide (PI) staining solution were from BD Biosciences. IL-3, IL-6, stem cell factor, and hTGF-β were from R&D Systems, and IL-12 and IL-4 were from PeproTech. His-tagged human USP15 recombinant protein was from Enzo Life Sciences. His-USP15 and His-USP15 C269A proteins were also produced by transfecting HEK293 cells with pcDNA-Xpress-His-USP15 and pcDNA-Xpress-His-USP15 C269A and purified using His60 Ni Resin (Takara). HLI373 was from TOCRIS, and Nutlin-3 was from Cayman [(−)-Nutlin-3]. CHX, Phorbol 12-myristate 13-acetate (PMA), and ionomycin were from Sigma-Aldrich.

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Zou Q., Jin J., Hu H., Li H.S., Romano S., Xiao Y., Nakaya M., Zhou X., Cheng X., Yang P., Lozano G., Zhu C., Watowich S.S., Ullrich S.E, & Sun S.C. (2014). USP15 stabilizes MDM2 to mediate cancer cell survival and inhibit antitumor T cell responses. Nature immunology, 15(6), 562-570.

Publication 2014

APC-Annexin V FITC-Annexin V Propidium Iodide IL-3 IL-6 stem cell factor hTGF-β IL-12 IL-4 His-tagged human USP15 recombinant protein His60 Ni Resin HLI373 Nutlin-3 Phorbol 12-myristate 13-acetate (PMA), ionomycin

Annexin v Antibodies Cayman Cell Fitc Flow cytometry Hek293 cells Hli373 Human usp15 protein Il 12 Immunoblot Ionomycin Nutlin 3 Phorbol myristate acetate Propidium iodide Proteins Resin Stem cell factor

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

His-tagged human USP15 recombinant protein
His-USP15 and His-USP15 C269A proteins
HLI373
Nutlin-3
Phorbol 12-myristate 13-acetate (PMA)
Ionomycin

dependent variables

Cell stimulation
Immunoblot
Flow cytometry

control variables

Antibodies used in this study (listed in Supplementary Table 4)
APC-Annexin V, FITC-Annexin V and Propidium Iodide (PI) staining solution
IL-3, IL-6, stem cell factor, and hTGF-β
IL-12 and IL-4

controls

Positive control: His-USP15 and His-USP15 C269A proteins produced by transfecting HEK293 cells
Negative control: Not explicitly mentioned

Annotations

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