We chose to investigate in-depth the three most abundant alternative TAPBPR transcripts, which were found multiple times in a number of different cell lines (Table 1). We focused on TAPBPR β, γ and δ, comparing various properties of these alternative transcripts with TAPBPR α. First, to determine if the three alternative TAPBPR transcripts produced an alternative TAPBPR protein product, we cloned cDNA from the TAPBPR β, γ, δ, transcripts into lentiviral expression vectors containing a bi-cistronic GFP reporter. The constructs were then transduced into HeLa cells, which do not express TAPBPR unless treated with IFN-γ. Flow cytometry for the GFP reporter revealed good transduction efficiency for all TAPBPR containing vectors into HeLa (α = 93%, β = 88%, γ = 93%, δ = 91%) (α and β are shown in Fig. 4a). The mean fluorescence intensity of GFP was comparable in all cell lines ranging from mean fluorescence intensity of 243 for TAPBPR α transduced cells to an mean fluorescence intensity of 270 for TAPBPR β transduced cells (Fig. 4b). To detect transduced TAPBPR protein, Western blotting was performed for TAPBPR using an antibody raised against amino acid 23–122 of TAPBPR, a region encoded in all of the TAPBPR isoforms. This revealed significant protein expression of TAPBPR α, β and γ (Fig. 4c). However, we failed to detect significant levels of the TAPBPR δ protein when expressed in HeLa although a faint band was observed in Western blot experiments (Fig. 4c). As TAPBPR δ does not contain a TMD, one reasonable explanation for the lack of significant detection in cell lysates by Western blot analysis was that this protein product was secreted by the HeLa cells. To determine if this was the case, HeLa cells transfected with the various isoforms of TAPBPR were cultured overnight in serum-free media. This cell culture medium was then collected, concentrated, separated by SDS–PAGE, and then Western blotting for TAPBPR was performed. However, we failed to detect any secreted TAPBPR δ, or any of the other TAPBPR isoforms, in the cell culture supernatants (Fig. 4d). Therefore, the lack of detection of TAPBPR in cells does not appear to be the result of protein secretion. As the stop codon encoded in TAPBPR δ is more than 50 nucleotides upstream of the 3′ exon–exon junction, it is likely that this transcript is subject to nonsense-mediated mRNA decay and so is not translated into a protein product in any significant quantities.[13, 14]
Figure 4. Protein translation from the alternative TAPBPR transcripts. (a) Cytofluorometric analysis for green fluorescent protein (GFP) expression from HeLa cells (black line histogram) and HeLa transduced with a lentiviral expression vector with a bi-cistronic GFP reporter and either TAPBPR α (grey-filled histogram) or TAPBPR β (black dotted line). (b) Bar graph of mean fluorescence intensity (MFI) of GFP on the full panel of TAPBPR isoforms from three independent experiments (Error bars: ± SEM). (c) Western blot analysis for TAPBPR (using mouse anti-TAPBPR; Abcam) or calnexin as a loading control from non-transduced HeLa (−) or HeLa stably transduced with lentiviral expression construct containing TAPBPR α, β, γ, δ. (d) Western blot analysis for TAPBPR from precipitated culture supernatants of non-transduced HeLa (−) or HeLa stably tranduced with TAPBPR α, β, γ or δ expression constructs. Western blot for TAPBPR on cellular lysates from the same cells is included as a positive control. (e) Western blot analysis for TAPBPR (using mouse anti-TAPBPR; Abcam) or calnexin as a loading control on HeLa and THP-1 cells ± IFN-γ treatment. (f) TAPBPR was isolated by immunoprecipitation (using polyclonal antiserum R039) from U937, THP-1 and KG-1 cells. Western blot analysis was performed for TAPBPR using mouse anti-TAPBPR. Western blotting with calnexin on lysates is included as a loading control.
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