Figure 5.
Deoxynojirimycin-treatment enhances the flux of YFP-PrP* through the RESET pathway, the binding of glycoproteins to CNX, and the release of YFP-PrP* from CNX. (A) Time-lapse imaging of representative YFP-PrP* and Cerulean (CER)-GalT NRK cells after treatment with 2.5 mM deoxynojirimycin alone (DNJ) or with 50 μg/ml cycloheximide (DNJ+CHX), or with 0.5 μM anisomycin (DNJ+An). CER-GalT is a Golgi marker. (B) Time-lapse imaging of representative YFP-PrP* NRK cells that were treated with 2.5 mM DNJ, DNJ+CHX, DNJ, DNJ+An, 0.5 mM dithiothreitol (DTT), or 1 μM thapsigargin (TG). (C) Representative western blots depicting GFP-column purifications from N2a cell lysates of untransfected (parent) cells or GFP-CNX-transfected cells 48 h after transient transfection. Blots were probed with anti-CNX (αCNX) antibody to detect GFP-CNX (green arrow) or endogenous CNX (red arrowhead) that co-eluted with GFP-CNX or probed with wheat germ agglutinin to detect general glycosylated proteins that co-eluted with GFP-CNX. Input “I” and eluate “E.” Cells were untreated (“U”) or treated with DNJ, TG, and DTT for 30 min, as indicated, prior to lysis. The experiment was performed in triplicate with similar results. (D and E) Representative western blots depicting flag-column purifications from N2a cell lysates of untransfected (parent) cells or transfected cells ∼72 h after transient transfection with (D) SNAPflag-CNX and CD3δ-CFP or (E) SNAPflag-CNX and YFP-PrP*. Blots were probed with anti-CNX (αCNX) antibody to detect SNAPflag-CNX (green arrow) or endogenous CNX (red arrowhead) that coeluted with SNAPflag-CNX and with anti-GFP antibody to probe for (D) CD3δ-CFP or (E) YFP-PrP* that co-eluted with SNAPflag-CNX. Input “I” and eluate “E.” Cells were untreated “U” or treated with DNJ for 15 m, as indicated, prior to lysis. Experiments were performed in triplicate. Integrated densitometry measurements were made of the αGFP bands. Quantification of the relative pixel density of the αGFP bands that co-purified with SNAPflag-CNX was made by first creating a rectangular bounding box or region of interest (ROI) that enclosed the larger eluate band. For D, the larger αGFP band was in the +DNJ eluate lane. For E, the larger αGFP band was in the untreated “U” eluate lane. Next, for D and E respectively, the ROI that enclosed the larger αGFP eluate band was dragged to enclose and measure the integrated density of αGFP eluate band untreated “U” and "+DNJ" co-eluates, and integrated densities within the bounding box were measured. Finally, the eluate αGFP bands in the "U" and "+DNJ" lanes were background subtracted and normalized against the “U” eluate band to obtain the relative pixel densities. These results are representative of three independent experiments (n = 3, biological replicates) ± SD. Source data are available for this figure: SourceData F5.

Deoxynojirimycin-treatment enhances the flux of YFP-PrP* through the RESET pathway, the binding of glycoproteins to CNX, and the release of YFP-PrP* from CNX. (A) Time-lapse imaging of representative YFP-PrP* and Cerulean (CER)-GalT NRK cells after treatment with 2.5 mM deoxynojirimycin alone (DNJ) or with 50 μg/ml cycloheximide (DNJ+CHX), or with 0.5 μM anisomycin (DNJ+An). CER-GalT is a Golgi marker. (B) Time-lapse imaging of representative YFP-PrP* NRK cells that were treated with 2.5 mM DNJ, DNJ+CHX, DNJ, DNJ+An, 0.5 mM dithiothreitol (DTT), or 1 μM thapsigargin (TG). (C) Representative western blots depicting GFP-column purifications from N2a cell lysates of untransfected (parent) cells or GFP-CNX-transfected cells 48 h after transient transfection. Blots were probed with anti-CNX (αCNX) antibody to detect GFP-CNX (green arrow) or endogenous CNX (red arrowhead) that co-eluted with GFP-CNX or probed with wheat germ agglutinin to detect general glycosylated proteins that co-eluted with GFP-CNX. Input “I” and eluate “E.” Cells were untreated (“U”) or treated with DNJ, TG, and DTT for 30 min, as indicated, prior to lysis. The experiment was performed in triplicate with similar results. (D and E) Representative western blots depicting flag-column purifications from N2a cell lysates of untransfected (parent) cells or transfected cells ∼72 h after transient transfection with (D) SNAPflag-CNX and CD3δ-CFP or (E) SNAPflag-CNX and YFP-PrP*. Blots were probed with anti-CNX (αCNX) antibody to detect SNAPflag-CNX (green arrow) or endogenous CNX (red arrowhead) that coeluted with SNAPflag-CNX and with anti-GFP antibody to probe for (D) CD3δ-CFP or (E) YFP-PrP* that co-eluted with SNAPflag-CNX. Input “I” and eluate “E.” Cells were untreated “U” or treated with DNJ for 15 m, as indicated, prior to lysis. Experiments were performed in triplicate. Integrated densitometry measurements were made of the αGFP bands. Quantification of the relative pixel density of the αGFP bands that co-purified with SNAPflag-CNX was made by first creating a rectangular bounding box or region of interest (ROI) that enclosed the larger eluate band. For D, the larger αGFP band was in the +DNJ eluate lane. For E, the larger αGFP band was in the untreated “U” eluate lane. Next, for D and E respectively, the ROI that enclosed the larger αGFP eluate band was dragged to enclose and measure the integrated density of αGFP eluate band untreated “U” and "+DNJ" co-eluates, and integrated densities within the bounding box were measured. Finally, the eluate αGFP bands in the "U" and "+DNJ" lanes were background subtracted and normalized against the “U” eluate band to obtain the relative pixel densities. These results are representative of three independent experiments (n = 3, biological replicates) ± SD. Source data are available for this figure: SourceData F5.

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