The expression of each transgene was also analyzed using western blot (Figure 1P-S). Blots were prepared in quadruplicate and were probed with antibodies against either the HA tag (Figure 1P), GFP (Figure 1Q), 2A (Figure 1R) or glyceraldehydes 3-phosphate dehydrogenase (GAPDH) (Figure 1S). Examination of the blots probed with either the HA tag or the GFP antibody showed that each antibody specifically recognized either mCherH or GFP, respectively, in protein samples prepared from cultures expressing either the EF1-GFP-EF1-mCherH or the EF1-GFP-W- EF1-mCherH transgene. In samples isolated from cultures expressing the EF1-GFP-2A-mCherH transgene, these antibodies detected both cleaved and uncleaved GFP-2A-mCherH. Cleaved GFP-2A was also readily detected using the 2A antibody, which has been reported to bind to 2A in the context of the cleaved peptide but not in the context of uncleaved peptide [28]. The relative amounts of GFP and mCherH produced from each transgene were determined using a scanning densitometer and the GFP and mCherH values were normalized to the highest value obtained for each protein, respectively (Figure 1T). Uncleaved GFP-2A-mCherH was not included in the determination of the amount of GFP and mCherH produced in cells expressing the EF1-GFP-2A-mCherH transgene. An analyses of variance re- vealed significant differences between transgenes in terms of GFP expression (F(2,6) = 8.92, p = 0.016); post hoc analyses using Tukey tests showed that the relative amount of GFP produced by the EF1-GFP-EF1-mCherH transgene (M = 0.53, SD = 0.17) was Lentiviral Delivery of Proteins Using 2A Peptides significantly lower than that produced from either the EF1-GFP- WPRE-EF1-mCherH (M = 0.94, SD = 0.10) or the EF1-GFP-2A- mCherH (M = 0.88, SD = 0.11) transgene. No differences were observed in relative mCherH expression between transgenes (F(2,6) = 3.515, p = 0.098). Estimates of the cleavage efficiency of GFP-2A-mCherH were calculated by expressing the intensities of the cleaved mCherH (Figure 1P) or GFP-2A (Figure 1Q) protein bands as a percentage of total HA-tag or GFP immunoreactivity, respectively. The estimates produced from these calculations were 69%64 (GFP, M6SD; n = 3) and 77%63 (HA, M6SD; n = 3). Thus, conservatively the cleavage efficiency of the GFP-2A- mCherH protein expressed in the HEK 293FT cells was approximately 73% (Figure 1U). Expression of the Packaged Viral Bicistronic Transgenes in 293FT Cell Cultures This series of experiments was carried out to determine if viral delivery of the bicistronic transgenes alters the expression characteristics of the transgenes. HEK 293FT cell cultures were infected with lentiviruses that carried one of the three bicistronic transgenes and expression of GFP and mCherH were analyzed using fluorescent microscopy and western blot. Comparisons of cultures infected with these viruses showed that the levels of GFP produced from the EF1-GFP-EF1-mCherH (Figure 2A) and EF1- GFP-W-EF1-mCherH (Figure 2E) transgenes were significantly lower than those produced from the EF1-GFP-2A-mCherH Figure 2. Expression characteristics of lentiviruses carrying the bicistronic transgenes in transduced HEK 293FT cells. (A–D), (E–H) and (I–L) are representative images of HEK 293FT cell cultures transduced with the indicated lentiviruses 96 h post-transduction. Image exposure times: GFP images in panels A, E, and I = 500 ms; mCher images in panels B, F, and J = 180 ms; Hoechst images in panels D, H, and L = 5 ms. Scale bar in A = 100 mm. (M–P) Western blot analyses of the expression of the GFP and mCherH reporter proteins in HEK293FT cells transduced with the lentiviruses. Three replicate HEK 293FT cultures were analyzed for each virus and one culture was analyzed for each of the control viruses, EF1- mCherH (CH) and EF1-GFP (GFP). Four identical western blots containing the protein samples were generated and each was probed with an antibody recognizing either hemagglutinin (M), GFP (N), 2A (O), or GAPDH (P). GAPDH was used as a protein loading control. (Q) Densitometric analyses of GFP and mCherH protein levels produced from each transgene. Normalized mean GFP and mCherH values were compared using paired T-tests and significant differences between samples (**p,.01, ***p,.001) are indicated with brackets. (R) The cleavage efficiency of GFP-2A-mCherH was estimated by expressing the intensities of the cleaved GFP-2A (N) or mCherH (M) protein bands as a percentage of total GFP or mCherH immunoreactivity (intensity of the cleaved plus uncleaved protein bands), respectively. Each estimate is the mean6SD of three samples. doi:10.1371/journal.pone.0020553.g002 PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20553