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. 2010 Oct 7;6(10):e1001150. doi: 10.1371/journal.pgen.1001150

Figure 2. Detection of ORF1p and ORF2p from engineered L1 constructs.

Figure 2

A. Representative results from western blot analyses: Whole cell lysates derived from untransfected HeLa cells or HeLa cells transfected with the indicated L1 expression constructs were subjected to western blot analyses. Top panels: western blots conducted with anti-TAP antibodies (αTAP; left side) or anti-HA antibodies (αHA; right side) to detect epitope-tagged ORF2p. Middle panels: western blots conducted with anti-T7 antibodies (αT7) to detect epitope-tagged ORF1p. Lower panels: western blots conducted with anti-tubulin antibodies (αTubulin) served as a loading control. Molecular weight standards (Invitrogen, left side, and New England Biolabs, right side) are listed at the left of each series of gels. B. Protein detection specificity: Whole cell lysates derived from HeLa cells transfected with the indicated constructs were subjected to western blot analyses using antibodies against either endogenous ORF1p (αORF1) or endogenous ORF2p (αORF2). TAP-tagged ORF2p also was detected using an anti-TAP antibody. T7-tagged ORF1p also was detected with an anti-T7 antibody. Tubulin was detected using an anti-tubulin (αTubulin) antibody and served as a loading control. Molecular weight standards (Invitrogen) are listed at the left of each series of gels. C. RT-PCR analyses: RT-PCR reactions using RNAs isolated from whole cell lysates derived from transfected cells revealed that L1 RNA was expressed from each of the constructs. GAPDH mRNA detection was used to assess the quality of the RNA preparations and as a loading control. Reactions without template (PCR control) or reverse transcriptase (RT control) were used as negative controls. DNA size markers (Invitrogen) are indicated at the left of the gel. Colored cartoons of the constructs used in the experiments are indicated next to their respective names. The black lines indicate the 5′ and 3′ UTRs. The green and red boxes indicate ORF1 and ORF2p respectively. When present, epitope tags are indicated. All constructs contain the mneoI retrotransposition indicator cassette.