Figure 1.
ER localization of the preTCR complex in reconstituted HEK cells. (A) Schematic showing the constituent protein chains of the αβTCR and preTCR complexes within the plasma membrane. (B) HEK cells were transfected with constructs to express the complete αβTCR or preTCR complexes. Raw flow cytometry plots show the correlation between receptor expression (measured by TCR β-GFP) and surface staining (anti-TCRβ-AF647). (C) Quantification of multiple datasets derived from B, where bounding area around data points shows mean ± SEM (n = 3), with P < 0.01 at all expression levels. (D) HEK cells were cotransfected with constructs for αβTCR or preTCR (GFP fused to TCRβ), along with ER-localized BFP, plasma membrane–localized mCherry, and nuclear-localized iRFP fluorophores. Representative images are shown, and colored boxes denote protein representation in the overlay image. Scale bar, 10 μm. (E) HEK cells expressing the αβTCR or preTCR were lysed and the complexes purified through the GFP fused to TCRβ chains. Isolated complexes were incubated with either EndoH or PNGaseF enzymes to remove defined glycosylation moieties. Samples were then subjected to Western blot analysis, probing for the HA epitope expressed at the cleaved N-terminus of TCR α or pTa. The blot shown is representative of three replicates. (F) HEK cells expressing the denoted receptor were treated with CHX overnight or vehicle control (DMSO) before purifying TCRβ as in A. Samples were blotted for GFP (fused to TCRβ) and actin as a loading control. (G) Quantification of Western blots as in F, with integrated band density for each receptor normalized to control. Datapoints show mean ± SEM (n = 3); asterisks indicate P < 0.01 when comparing αβTCR and preTCR datasets in the presence of CHX. A two-tailed, two-sided t test was used for all statistical analyses. Source data are available for this figure: SourceData F1.

ER localization of the preTCR complex in reconstituted HEK cells. (A) Schematic showing the constituent protein chains of the αβTCR and preTCR complexes within the plasma membrane. (B) HEK cells were transfected with constructs to express the complete αβTCR or preTCR complexes. Raw flow cytometry plots show the correlation between receptor expression (measured by TCR β-GFP) and surface staining (anti-TCRβ-AF647). (C) Quantification of multiple datasets derived from B, where bounding area around data points shows mean ± SEM (n = 3), with P < 0.01 at all expression levels. (D) HEK cells were cotransfected with constructs for αβTCR or preTCR (GFP fused to TCRβ), along with ER-localized BFP, plasma membrane–localized mCherry, and nuclear-localized iRFP fluorophores. Representative images are shown, and colored boxes denote protein representation in the overlay image. Scale bar, 10 μm. (E) HEK cells expressing the αβTCR or preTCR were lysed and the complexes purified through the GFP fused to TCRβ chains. Isolated complexes were incubated with either EndoH or PNGaseF enzymes to remove defined glycosylation moieties. Samples were then subjected to Western blot analysis, probing for the HA epitope expressed at the cleaved N-terminus of TCR α or pTa. The blot shown is representative of three replicates. (F) HEK cells expressing the denoted receptor were treated with CHX overnight or vehicle control (DMSO) before purifying TCRβ as in A. Samples were blotted for GFP (fused to TCRβ) and actin as a loading control. (G) Quantification of Western blots as in F, with integrated band density for each receptor normalized to control. Datapoints show mean ± SEM (n = 3); asterisks indicate P < 0.01 when comparing αβTCR and preTCR datasets in the presence of CHX. A two-tailed, two-sided t test was used for all statistical analyses. Source data are available for this figure: SourceData F1.

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