Figure 7.
New expression of CNX-binding N-linked glycoproteins triggers RESET of PrP*. (A) Western blots depicting GFP column purifications from parental, untransfected N2a cells (parent), or N2a cells 12 h after transient transfection with ER-targeted CFP-KDEL or CD3δ-CFP. Blots were probed with anti-GFP antibodies to detect CFP-KDEL or CD3δ-CFP and with anti-CNX antibody to detect endogenous CNX. Blue asterisk and red arrowhead placed next to the GFP-blot align with full-length CD3δ-CFP and CFP-KDEL, respectively. Input “I,” eluate “E.” (B and C) Time-lapse imaging of representative NRK cells stably expressing YFP-PrP* and the Golgi marker, FusionRed-SiT (FusRed-SiT), and transiently transfected with (B) ER-targeted CFP (CFP-KDEL) or (C) CD3δ-CFP. Image collection was started 2 h after transient transfection. Scale bar, 10 μm. (D–F) Western blots depicting GFP column purifications from parental, untransfected N2a cells (parent) or N2a cells 12 h after transiently transfecting cells with (D) CD3δ-CFP or Cerulean-Thy1, (E) Cerulean-IL-6 or Cerulean-vIL-6, and (F) Cerulean-SARS CoV-2 Spike Glycoprotein “Cer-SARS2 Spike,” and probed with anti-GFP antibody or anti-CNX antibody. Input “I” and eluate “E.” (G–J) Time-lapse imaging of YFP-PrP* NRK cells that were transiently transfected with Cerulean-tagged N-linked glycoproteins that were reported to be upregulated during inflammatory conditions, including (G) Thy1, (H) IL-6, and during a viral infection (I) vIL-6 and (J) SARS2 Spike. For G–I, the fields of view include one cell that was transfected with the Cerulean-tagged N-linked glycoprotein and one cell that remained untransfected. The arrowheads point to cells which were newly expressing the transfected N-linked glycoproteins. For J, cells stably expressed Golgi marker, FusRed-SiT. For G–J, image collection was started 2 h after addition of transfection reagent and plasmids. Scale bar is 10 um. Source data are available for this figure: SourceData F7.

New expression of CNX-binding N-linked glycoproteins triggers RESET of PrP*. (A) Western blots depicting GFP column purifications from parental, untransfected N2a cells (parent), or N2a cells 12 h after transient transfection with ER-targeted CFP-KDEL or CD3δ-CFP. Blots were probed with anti-GFP antibodies to detect CFP-KDEL or CD3δ-CFP and with anti-CNX antibody to detect endogenous CNX. Blue asterisk and red arrowhead placed next to the GFP-blot align with full-length CD3δ-CFP and CFP-KDEL, respectively. Input “I,” eluate “E.” (B and C) Time-lapse imaging of representative NRK cells stably expressing YFP-PrP* and the Golgi marker, FusionRed-SiT (FusRed-SiT), and transiently transfected with (B) ER-targeted CFP (CFP-KDEL) or (C) CD3δ-CFP. Image collection was started 2 h after transient transfection. Scale bar, 10 μm. (D–F) Western blots depicting GFP column purifications from parental, untransfected N2a cells (parent) or N2a cells 12 h after transiently transfecting cells with (D) CD3δ-CFP or Cerulean-Thy1, (E) Cerulean-IL-6 or Cerulean-vIL-6, and (F) Cerulean-SARS CoV-2 Spike Glycoprotein “Cer-SARS2 Spike,” and probed with anti-GFP antibody or anti-CNX antibody. Input “I” and eluate “E.” (G–J) Time-lapse imaging of YFP-PrP* NRK cells that were transiently transfected with Cerulean-tagged N-linked glycoproteins that were reported to be upregulated during inflammatory conditions, including (G) Thy1, (H) IL-6, and during a viral infection (I) vIL-6 and (J) SARS2 Spike. For G–I, the fields of view include one cell that was transfected with the Cerulean-tagged N-linked glycoprotein and one cell that remained untransfected. The arrowheads point to cells which were newly expressing the transfected N-linked glycoproteins. For J, cells stably expressed Golgi marker, FusRed-SiT. For G–J, image collection was started 2 h after addition of transfection reagent and plasmids. Scale bar is 10 um. Source data are available for this figure: SourceData F7.

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