GFPdnLMP1 expression is lost from EμLMP1 transgenic B-cell lymphoma cultures. Transgenic EμLMP1 cell lines 39.415 and 3959.48, along with an EBV negative Akata cell line sub clone (AK31), transfected with pGFP or pGFPdnLMP1 were assayed for transfectant expression. (A) Protein extract from 5 × 105 39.415 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected after completion of selection at 3 weeks post transfection (post-tx) and then at weekly intervals (maintaining G418 selective pressure). (B) Bright field (left panel) and green fluorescence (right panel) visualized in pGFPdnLMP1 (top panel) or pGFP (bottom panel) transfected 39.415 cells at 3 weeks post transfection. (C) 40 μg of protein extract from 3959.48 and control AK31 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected at 2, 5 and 21 days post transfection (post-tx) for 3959.48 cells and at 12 weeks post-tx for AK31 cells (all under G418 selection). (D) At four weeks post pGFP or pGFPdnLMP1 transfection 3959.48 cells stained with propidium iodide (viable cells exclude staining, apoptotic cells stain) were analysed by flow cytometry, gating on GFP positive fluorescent cells only. Histograms show y axis (cell counts) and x axis (FL2-H, PI staining). The percentage of PI positive cells (of the GFP positive population) is indicated.