SOCS1 Regulation in HEK293 Cells

HEK293 cells were maintained in complete DMEM. Cells were transiently transfected with pCMV6-AC-GFP Vector (OriGene), which expresses SOCS1 (mouse; available from GenBank under accession no. NM_009896.2) driven by the constitutive CMV promoter, using Lipofectamine 2000 reagent according to the manufacturer’s protocol. Cysteine mutations (C147S, C179S, C43S, C78S and C112S) were introduced in SOCS1 ORF (GenScript USA Inc.). HEK293 cells were stimulated with a 15 min pulse of 20 ng/ml IL-1β (Prospec Inc.) and treated with 10 µM DEA NONOate (DEANO) for 1 h (Alexis Biochemicals). Diethylamine NO (DEANO) is a member of the NONOate class NO donors. At 37°C, pH 7.4, it generates NO spontaneously with a half-life of 2.1 min (Schödel et al., 2009 (link)). Total SOCS1 and corresponding p65 protein level was also determined in the presence and absence of 50 µM MG132 (Sigma-Aldrich) to inhibit proteasomal lysis.

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Baig M.S., Zaichick S.V., Mao M., de Abreu A.L., Bakhshi F.R., Hart P.C., Saqib U., Deng J., Chatterjee S., Block M.L., Vogel S.M., Malik A.B., Consolaro M.E., Christman J.W., Minshall R.D., Gantner B.N, & Bonini M.G. (2015). NOS1-derived nitric oxide promotes NF-κB transcriptional activity through inhibition of suppressor of cytokine signaling-1. The Journal of Experimental Medicine, 212(10), 1725-1738.

Publication 2015

pCMV6-AC-GFP Vector IL-1β MG132

Cells Cysteine Dea nonoate Diethylamine Donors Hek293 cells Il 1β Il 20 Inhibit Lipofectamine 2000 Mg132 Mouse Mutations Prospec Proteasomal Protein Pulse Vector

Corresponding Organization : Indiana University – Purdue University Indianapolis

Other organizations : Universidade Estadual de Maringá, University of South Carolina

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Variable analysis

independent variables

Transfection of HEK293 cells with pCMV6-AC-GFP Vector (OriGene) expressing SOCS1 (mouse)
Introduction of cysteine mutations (C147S, C179S, C43S, C78S and C112S) in SOCS1 ORF
Stimulation of HEK293 cells with a 15 min pulse of 20 ng/ml IL-1β (Prospec Inc.)
Treatment of HEK293 cells with 10 µM DEA NONOate (DEANO) for 1 h (Alexis Biochemicals)

dependent variables

Total SOCS1 protein level
P65 protein level

control variables

Maintenance of HEK293 cells in complete DMEM
Use of Lipofectamine 2000 reagent for transfection according to the manufacturer's protocol
Treatment with 50 µM MG132 (Sigma-Aldrich) to inhibit proteasomal lysis

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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