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. 2020 Jun 19;11:3106. doi: 10.1038/s41467-020-16939-6

Fig. 1. NMD-polypeptides are targeted to the aggresome.

Fig. 1

a The schematic diagram of NMD reporter mRNAs: FLAG-GPx1-Norm and -Ter [harboring either a normal termination codon or premature termination codon (PTC), respectively]. The regions encoding three tandem repeats of the FLAG tag (3xFLAG) and GPx1 are specified by black and dark gray boxes, respectively. AUG, translation initiation codon; UAA, translation termination codon. b Immunostaining of CFTR-∆F508 (green) and either FLAG-GPx1-Norm or -Ter polypeptides (red). HeLa cells stably expressing CFTR-ΔF508 were transiently transfected with a plasmid expressing either 3xFLAG-GPx1-Norm or -Ter. The cells were treated with either dimethyl sulfoxide (DMSO) or MG132 for 12 h before immunostaining. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). A magnified view of the white boxed area is provided in the lower right corner of each image. c Immunostaining of a known aggresomal marker (γ-tubulin; green) and the FLAG-GPx1-Ter polypeptides (red). d Immunostaining of CFTR-∆F508 (green) and the FLAG-GPx1-Ter polypeptides (red) after downregulation of a component of the CED complex (CTIF, eEF1A1, or DCTN1). HeLa cells stably expressing CFTR-ΔF508 were transfected with the indicated siRNA. Two days later, the cells were retransfected with a plasmid expressing FLAG-GPx1-Ter. The cells were treated with either DMSO or MG132 for 12 h before the immunostaining. Immunostaining images in each panel are representative of at least two independently processed biological replicates (n = 3 for (b) and (c) and n = 2 for (d). Scale bar, 10 μm.