Journal: Molecular & Cellular Proteomics : MCP
Article Title: HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation
doi: 10.1016/j.mcpro.2022.100488
Figure Lengend Snippet: Effect of NPM1 Ser-125 phosphorylation on HIV-1 transcription . A and B , effect of expression of NPM1 and its phosphorylation mutants (S125A and S125D) on basal (panel A ) and Tat-mediated HIV-1 transcription (panel B ). HEK293T cells were transfected with vectors expressing the indicated GFP-tagged NPM1 mutants and cotransfected with vectors expressing HIV-1 LTR-luciferase reporter (panel A ) or HIV-1 LTR-luciferase reporter and FLAG-tagged Tat expression vector (panel B ). Luciferase activity was analyzed using Luclite plus Reporter Gene Assay and normalized to GFP intensity. All data were shown in triplicates as transcription activation fold related to the WT NPM1. C , effect of NPM1 Ser-125 phosphorylation on the interaction with Tat protein. GFP-tagged NPM1 (WT, S125A, and S125D) and Flag-tagged HIV-1 Tat were expressed in 293T cells. Tat was precipitated with anti-Flag antibody, resolved on SDS-PAGE, and immunoblotted with anti-GFP to detect NPM1 and anti-Flag antibodies to detect Tat. Lane 1, IgG control; Lane 2, No Tat; Lane 3, Tat+NPM1 WT; Lane 4, Tat+NPM1 S125A; Lane 5, Tat+NPM1 S125D. D – G , quantification of the coimmunoprecipitation data from three independent experiments. NPM1’s 62 kDa and 250 kDa isoforms were normalized to NPM1 and Tat input, respectively. Asterisks indicate p < 0.05 (∗) and p < 0.01 (∗∗). HIV-1, human immunodeficiency virus-1; LTR, long terminal repeat; NPM1, nucleophosmin.
Article Snippet: Cells were cultured 48 h post-transfection, and luciferase activity was analyzed using Luclite plus Reporter Gene Assay (PerkinElmer) measured by Glo-Max Microplate Multimode reader (Promega).
Techniques: Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Reporter Gene Assay, Activation Assay, SDS Page, Control, Virus
