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. 2014 May 6;22(6):1084–1095. doi: 10.1038/mt.2014.52

Figure 2.

Figure 2

RhTRIM5α accumulates much more readily than huTRIM5α in primary human T cells. (a) CD3/28-activated CD4 T cells were transduced with lentiviral vectors encoding TRIM5α isoforms linked to green fluorescent protein (GFP) via a 2A sequence (top) or fused to mCherry (bottom). GFP and mCherry expression was measured by flow cytometry after 8 days of culture (shown), and at other timepoints in replicate experiments (box-plot). (b) The relative abundance of each TRIM5α isoform mCherry was measured by quantitative revese transcription polymerase chain reaction. (c) Protein lysates were made from the cells shown in Figure 2a and blotted for TRIM5α (red) or ZAP-70 (green); relative expression of each TRIM5α isoform compared to untransduced T cells is shown below each sample (upper panel). Lysates from GFP-2A-TRIM5α transduced cells and controls were blotted separately for GFP (lower panel). (d) Immunoprecipitation of mCherry-TRIM proteins from transduced primary human CD4 T cells via monoclonal antibody to mCherry, followed by western blotting with anti-TRIM5α (left panel) and anti-mCherry (right panel) polyclonal antibodies. (e) Densitometry data are representative of at least four independent experiments.