The HB*-tagged UPF1, UPF2, and SMG5 constructs are functionally active in the NMD pathway.

<p><b>(A)</b> Schematic of the TCRβ ter68 NMD reporter construct stably integrated in HeLa Tet-Off cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.ref024" target="_blank">24</a>]. The TRE-tight promoter (thick black arrow), the transcription start site (thin arrow) and the SV40 polyadenylation site (black box with flag) are indicated. AUG denotes the beginning of the open reading frame (box), and its sites of premature and normal termination are marked by the premature termination codon (PTC) and the termination codon (TC), respectively. The two introns are depicted as kinked lines <b>(B)</b> Schematic representation of the tested protein constructs. The HA-tag followed by a Gly₁₆ spacer is shown as white box, BirA as orange box (with the R118G mutation denoted as white star) and bait factors as blue boxes <b>(C-E)</b> Knockdown of the indicated NMD factors was achieved by transiently transfecting the HeLa Tet-Off TCRβ ter68 cells with shRNA expressing plasmids and rescue of NMD activity was attempted by co-transfection of the expression plasmids encoding the indicated RNAi resistant (denoted ^R) constructs. Relative TCRβ ter68 RNA levels, normalized to ACTB mRNA, were measured by real-time qRT-PCR (left side) and quantified by the ΔΔCT method. The columns represent the average expression level of three independent biological replicates. Error bars mark the cumulated variation among the ΔΔCT values in the biological replicates. The levels of the indicated proteins were analyzed by western blotting (right side). (C) Knockdown of endogenous UPF1 can be rescued by expressing H16-UPF1^R, HB-UPF1^R or HB*-UPF1^R fusion proteins, (D) knockdown of endogenous UPF2 can be rescued by expressing H16-UPF2^R, HB-UPF2^R or HB*-UPF2^R fusion proteins, and (E) knockdown of endogenous SMG5 can be rescued by expressing H16-SMG5^R, HB-SMG5^R, and HB*-SMG5^R fusion proteins. Positions of recombinant and endogenous proteins on the blots are indicated on the left by white and black triangles, respectively.