Proteomic workflow and validation studies of the SILAC analysis of Jurkat-T cells expressing HIV-1 Tat.

<p><b>A.</b> Organellar proteomic workflow for the analysis of quantitative changes in Jurkat NTAP-Tat versus the Jurkat NTAP cells. <b>B.</b> Designed cassettes for NTAP and NTAP-Tat. GFP is cloned downstream the NTAP-Tat and NTAP genes and its translation is independently controlled by IRES. FACS analysis of the Jurkat NTAP-Tat and Jurkat NTAP showing a highly enriched population of polyclonal transduced cells. <b>C.</b> We transfected the Jurkat NTAP-Tat and Jurkat NTAP using 2 and 5 µg of pGL3-LTR plasmid. HIV-1 LTR luciferase reporter gene assay confirmed that the NTAP-Tat is functionally active. <b>D.</b> Validation of the subcellular fractionation from mixed Jurkat NTAP-Tat (R0K0) and Jurkat NTAP (R6K6) (1∶1). Each fraction (10 µg of protein per lane) was checked by Western-Blot using anti-nucleolin (C23), anti-fibrillarin, anti-α-tubulin and anti-PARP antibodies. The molecular weight (kDa) of each protein is indicated on the left. (Fractions: WC: whole cells, C: Cytoplasmic, N: Nuclear, Np: Nucleoplasmic and No: Nucleolar). <b>E.</b> Expression and subcellular distribution of NTAP-Tat in Jurkat T-cells using Western-Blot analysis. NTAP-Tat (36 kDa) was detected using anti-HIV-1 Tat antibody (ab43014, Abcam). For immunofluorescence analysis, Jurkat NTAP-Tat cells were stained for fibrillarin (green), HIV-1 Tat (red), and DAPI (Blue or in grey contrast on the top left panel). NTAP-Tat overlaps with fibrillarin in the nucleolar compartment. (Bar: 2 µm). <b>F.</b> GO biological processes and cellular components distribution of the nucleolar proteome of mixed cells. <b>G.</b> Distribution of the changes in protein abundance in the nucleolus of Jurkat T-cells upon HIV-1 Tat expression. Relative abundance is plotted as Log2 (SILAC ratios). Green denotes depletion, while red denotes enrichment.