Figure 1.
Cycloheximide inhibits steady-state ER-export and degradation of YFP-PrP*. (A) Time-lapse imaging of representative YFP-PrP* NRK or YFP-CD3δ NRK stable transfectants upon treatment with 50 μg/ml cycloheximide. Scale bar, 10 μm. (B and C) Representative western blots of cycloheximide-chase assays of (B) YFP-PrP* (probed with anti-GFP antibody) and Tmp21 or (C) YFP-CD3δ (probed with anti-GFP antibody) from stably transfected NRK cells. Cells were collected at the indicated time points after cycloheximide treatment. An equal number of stably transfected cells were seeded in a 12-well dish 24 h prior to cycloheximide treatment. Total input panels labeled “lysates” depict Bio-Rad Stain-free gel imaging system to visually confirm equal loading of total cellular lysates across the time points. Experiments were performed in triplicate. (D) Relative pixel densities of YFP-PrP* (black bars) and YFP-CD3δ (white bars) bands of the western blots from the cycloheximide-chase assays, as shown in representative examples in B and C. Western blots of cell lysates were probed with anti-GFP antibody to detect YFP-PrP* and YFP-CD3δ. Quantifications of the YFP-PrP* or YFP-CD3δ bands were performed by creating a rectangular bounding box or region of interest (ROI) that surrounded and enclosed the baseline t = 0 band and then dragging the same bounding box from lane to lane to measure the integrated density of the GFP bands from t = 0 through t = 5 h. Integrated density values were background subtracted and normalized against the loading controls. Loading control values were obtained by measuring the integrated density signal across each lysate lane, instead of individual bands. The normalized density values were measured against the t = 0 value to obtain the relative pixel densities, where t = 0 is normalized to 1. Bar graph represents the average relative pixel density of three independently performed experiments (n = 3, biological replicates). Error bars represent SD. (E) Autoradiograph of steady-state chase of YFP-PrP* NRK cells that were left untreated “U” or treated with cycloheximide “+CHX.” Arrowhead is positioned at the molecular weight of the YFP-PrP* band. Anti-GFP immunoprecipitations to capture the YFP-PrP* were performed on cell lysates from YFP-PrP* NRK cells that were radiolabeled 12 h with 35S-labeled cysteine and methionine and chased with non-radioactive medium without “U” (for untreated) or with cycloheximide “+CHX” for 0, 2, or 4 h. (F) Quantification of the relative pixel density of the YFP-PrP* bands from triplicate steady-state chases of YFP-PrP* isolated from YFP-PrP* NRK cells that were left untreated “U” (black bars) or treated with cycloheximide “+CHX” (white bars). Quantifications of the radiolabeled YFP-PrP* bands were performed by creating a rectangular bounding box or region of interest (ROI) that surrounded and enclosed the baseline t = 0 band, and then dragging the same bounding box to enclose and measure the integrated density of the t = 2 and t = 4 h bands. Integrated density values were background subtracted, normalized against the intensity across the corresponding lysate lanes. The normalized density values were measured against the t = 0 value to obtain the relative pixel densities where t = 0 is normalized to 1 for untreated or +cycloheximide “+CHX” samples. Bar graph represents the average relative pixel density of three independently performed experiments (n = 3, biological replicates). Error bars represent SD. (G) Time-lapse imaging of a representative stably transfected YFP-PrP* and Cerulean (CER)-GalT NRK cell that was treated with CHX. CER-GalT marks the Golgi. Insets are magnified for 0 and 30 m time points. YFP-PrP* and CER-GalT intensity plot profiles of the lines drawn across the 0 and 30 m images are shown as a solid green line (YFP-PrP*) and dashed red line (CER-GalT). Intensity units are arbitrary (A.U.). Scale bar in the main figure is 10 μm, while scale bars in the insets are 1 μm. (H) Time-lapse imaging of a representative YFP-PrP* NRK cell that was treated with 1 μM thapsigargin after 16 h of cycloheximide “+CHX 16 h pre-treatment” (left panel) or after no pretreatment (right panel). Source data are available for this figure: SourceData F1.

Cycloheximide inhibits steady-state ER-export and degradation of YFP-PrP*. (A) Time-lapse imaging of representative YFP-PrP* NRK or YFP-CD3δ NRK stable transfectants upon treatment with 50 μg/ml cycloheximide. Scale bar, 10 μm. (B and C) Representative western blots of cycloheximide-chase assays of (B) YFP-PrP* (probed with anti-GFP antibody) and Tmp21 or (C) YFP-CD3δ (probed with anti-GFP antibody) from stably transfected NRK cells. Cells were collected at the indicated time points after cycloheximide treatment. An equal number of stably transfected cells were seeded in a 12-well dish 24 h prior to cycloheximide treatment. Total input panels labeled “lysates” depict Bio-Rad Stain-free gel imaging system to visually confirm equal loading of total cellular lysates across the time points. Experiments were performed in triplicate. (D) Relative pixel densities of YFP-PrP* (black bars) and YFP-CD3δ (white bars) bands of the western blots from the cycloheximide-chase assays, as shown in representative examples in B and C. Western blots of cell lysates were probed with anti-GFP antibody to detect YFP-PrP* and YFP-CD3δ. Quantifications of the YFP-PrP* or YFP-CD3δ bands were performed by creating a rectangular bounding box or region of interest (ROI) that surrounded and enclosed the baseline t = 0 band and then dragging the same bounding box from lane to lane to measure the integrated density of the GFP bands from t = 0 through t = 5 h. Integrated density values were background subtracted and normalized against the loading controls. Loading control values were obtained by measuring the integrated density signal across each lysate lane, instead of individual bands. The normalized density values were measured against the t = 0 value to obtain the relative pixel densities, where t = 0 is normalized to 1. Bar graph represents the average relative pixel density of three independently performed experiments (n = 3, biological replicates). Error bars represent SD. (E) Autoradiograph of steady-state chase of YFP-PrP* NRK cells that were left untreated “U” or treated with cycloheximide “+CHX.” Arrowhead is positioned at the molecular weight of the YFP-PrP* band. Anti-GFP immunoprecipitations to capture the YFP-PrP* were performed on cell lysates from YFP-PrP* NRK cells that were radiolabeled 12 h with 35S-labeled cysteine and methionine and chased with non-radioactive medium without “U” (for untreated) or with cycloheximide “+CHX” for 0, 2, or 4 h. (F) Quantification of the relative pixel density of the YFP-PrP* bands from triplicate steady-state chases of YFP-PrP* isolated from YFP-PrP* NRK cells that were left untreated “U” (black bars) or treated with cycloheximide “+CHX” (white bars). Quantifications of the radiolabeled YFP-PrP* bands were performed by creating a rectangular bounding box or region of interest (ROI) that surrounded and enclosed the baseline t = 0 band, and then dragging the same bounding box to enclose and measure the integrated density of the t = 2 and t = 4 h bands. Integrated density values were background subtracted, normalized against the intensity across the corresponding lysate lanes. The normalized density values were measured against the t = 0 value to obtain the relative pixel densities where t = 0 is normalized to 1 for untreated or +cycloheximide “+CHX” samples. Bar graph represents the average relative pixel density of three independently performed experiments (n = 3, biological replicates). Error bars represent SD. (G) Time-lapse imaging of a representative stably transfected YFP-PrP* and Cerulean (CER)-GalT NRK cell that was treated with CHX. CER-GalT marks the Golgi. Insets are magnified for 0 and 30 m time points. YFP-PrP* and CER-GalT intensity plot profiles of the lines drawn across the 0 and 30 m images are shown as a solid green line (YFP-PrP*) and dashed red line (CER-GalT). Intensity units are arbitrary (A.U.). Scale bar in the main figure is 10 μm, while scale bars in the insets are 1 μm. (H) Time-lapse imaging of a representative YFP-PrP* NRK cell that was treated with 1 μM thapsigargin after 16 h of cycloheximide “+CHX 16 h pre-treatment” (left panel) or after no pretreatment (right panel). Source data are available for this figure: SourceData F1.

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