Candidate list of Vpr-interacting partners in macrophages.
Description
Candidate list of Vpr-interacting partners in MDMs from two independent experiments. PBMCs obtained from healthy blood donors were purified using Ficoll-Hypaque gradient centrifugation. CD4+ T cells or monocytes were isolated from PBMCs via negative selection with CD14-positive enrichment cocktail (StemCell Technologies). Briefly, MDMs were generated by stimulating monocytes with 50 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D) for 7 days.
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Steps to reproduce
MDMs generated from the monocytes isolated from 10 healthy donors were pretreated with VLP-Vpx and transduced with a lentiviral vector expressing FLAG-Vpr from NL4-3. After puromycin selection, cells (5 x 106) were treated with or without 1.5 M MG132 for 8–10 h and lysed using IP lysis buffer (50 mM Tris-HCl, pH 7.2, 50 mM NaCl, 1% NP-40, 1 mM EDTA, 2% glycerol, 1× protease inhibitor cocktails, cOmplete), and the cell lysates from five donors were pooled (then two groups were obtained from a total of 10 donors for subsequent experiments). The cell lysates were incubated on ice for 30 min and centrifuged at 14,000 x g for 10 min at 4°C. The supernatants were transferred to fresh tubes, and the pellets were mixed with cold IP lysis buffer before sonication, followed by a second round of centrifugation at 14,000 x g for 10 min at 4°C. The supernatants obtained from the two extraction steps were pooled and incubated with a mix of protein A and protein G Dynabeads, which had been pretreated overnight with an anti-FLAG antibody at 4°C. The IP products were washed with cold IP buffer and PBST 5–10 times (500 μL per wash) and subjected with LC-MS/MS analysis. The raw data were processed using Proteome Discoverer, and MS/MS spectra were used to search the reviewed Swiss-Prot human proteome database. Only peptides of at least six amino acids in length were considered. At least one unique peptide was required for protein identification.