Figure 9.
The levels of CNX-binding substrates are inversely correlated with the levels of ER-localized or CNX-bound YFP-PrP*. (A) Time traces of normalized total intensity of YFP-PrP* (green) and CD3δ-CFP (gray) in the ER of three individual cells (Cell 1–3) that were imaged every 15 min upon the induction of new CD3δ-CFP expression. Details for how the time traces were generated are provided in the Materials and methods section. (B) Montage of time-lapse for Cell 1 depicted in A showing YFP-PrP* (green), CD3δ-CFP (grayscale), and the Golgi marker, FusionRed-SiT (FusRed-SiT) (red). (C) Western blots depicting column purifications of endogenous CNX from lysates of YFP-PrP* NRK cells that were treated with 0, 1.25, or 2.50 µg/ml of puromycin for 30 min. Input “I” and eluate “E.” 24 h prior to column purification (described in Materials and methods), equal numbers of cells were seeded into 10-cm dishes and cells were processed identically, with the exception of the 30 m puromycin treatments. Blots were probed with anti-CNX “αCNX” antibody to detect endogenous CNX, anti-GFP “αGFP” antibody to detect YFP-PrP*, and anti-puromycin “αPuro” antibody to detect puromycinylated proteins products that co-eluted with CNX. (D) Plots of the relative pixel densities representing YFP-PrP* and puromycinylated-proteins that co-eluted with endogenous CNX. To quantify the relative amounts of the YFP-PrP* or puromycinylated-proteins that co-purified with CNX, we performed the following steps. The integrated densities of the YFP-PrP* or puromycinylated co-eluates for each of the 0, 1.25 and 2.50 µg/ml puromycin-treated samples were measured within a bounding box or region of interest (ROI) sized to enclose the YFP-PrP* band in the 0 ug/ml puromycin eluate “E” lane or all of the bands spanning the 2.5 µg/ml puromycin eluate “E” lane, respectively, and background subtracted. To address the variation in CNX pulled down from the different samples, integrated densities of co-eluted YFP-PrP* or puromycinylated-products were normalized against the integrated densities of the corresponding CNX bands to produce relative amounts of PrP or puromycinylated-proteins that were bound to CNX. Finally, to facilitate evaluation of the proportional changes between samples, values were further normalized such that CNX-bound YFP-PrP* in untreated “0” cells were normalized to 1, and CNX-bound puromycinylated proteins were normalized so that the entire range falls between 0 (for untreated sample) to 1 (for the 2.5 μg/ml puromycin-treated sample). The plotted relative integrated densities are representative of three independently performed experiments (n = 3, biological replicates) ± SD. The relative values of CNX-associated YFP-PrP* in 0, 1.25, and 2.50 µg/ml puromycin-treated cells were 1.00, 0.88 ±0.04 (SD), and 0.59 ±0.09 (SD), respectively. The relative values of total CNX-associated puromycinylated-proteins in 0, 1.25, and 2.50 µg/ml puromycin-treated cells were 0, 0.60 ±0.07, and 1, respectively. Source data are available for this figure: SourceData F9.

The levels of CNX-binding substrates are inversely correlated with the levels of ER-localized or CNX-bound YFP-PrP*. (A) Time traces of normalized total intensity of YFP-PrP* (green) and CD3δ-CFP (gray) in the ER of three individual cells (Cell 1–3) that were imaged every 15 min upon the induction of new CD3δ-CFP expression. Details for how the time traces were generated are provided in the Materials and methods section. (B) Montage of time-lapse for Cell 1 depicted in A showing YFP-PrP* (green), CD3δ-CFP (grayscale), and the Golgi marker, FusionRed-SiT (FusRed-SiT) (red). (C) Western blots depicting column purifications of endogenous CNX from lysates of YFP-PrP* NRK cells that were treated with 0, 1.25, or 2.50 µg/ml of puromycin for 30 min. Input “I” and eluate “E.” 24 h prior to column purification (described in Materials and methods), equal numbers of cells were seeded into 10-cm dishes and cells were processed identically, with the exception of the 30 m puromycin treatments. Blots were probed with anti-CNX “αCNX” antibody to detect endogenous CNX, anti-GFP “αGFP” antibody to detect YFP-PrP*, and anti-puromycin “αPuro” antibody to detect puromycinylated proteins products that co-eluted with CNX. (D) Plots of the relative pixel densities representing YFP-PrP* and puromycinylated-proteins that co-eluted with endogenous CNX. To quantify the relative amounts of the YFP-PrP* or puromycinylated-proteins that co-purified with CNX, we performed the following steps. The integrated densities of the YFP-PrP* or puromycinylated co-eluates for each of the 0, 1.25 and 2.50 µg/ml puromycin-treated samples were measured within a bounding box or region of interest (ROI) sized to enclose the YFP-PrP* band in the 0 ug/ml puromycin eluate “E” lane or all of the bands spanning the 2.5 µg/ml puromycin eluate “E” lane, respectively, and background subtracted. To address the variation in CNX pulled down from the different samples, integrated densities of co-eluted YFP-PrP* or puromycinylated-products were normalized against the integrated densities of the corresponding CNX bands to produce relative amounts of PrP or puromycinylated-proteins that were bound to CNX. Finally, to facilitate evaluation of the proportional changes between samples, values were further normalized such that CNX-bound YFP-PrP* in untreated “0” cells were normalized to 1, and CNX-bound puromycinylated proteins were normalized so that the entire range falls between 0 (for untreated sample) to 1 (for the 2.5 μg/ml puromycin-treated sample). The plotted relative integrated densities are representative of three independently performed experiments (n = 3, biological replicates) ± SD. The relative values of CNX-associated YFP-PrP* in 0, 1.25, and 2.50 µg/ml puromycin-treated cells were 1.00, 0.88 ±0.04 (SD), and 0.59 ±0.09 (SD), respectively. The relative values of total CNX-associated puromycinylated-proteins in 0, 1.25, and 2.50 µg/ml puromycin-treated cells were 0, 0.60 ±0.07, and 1, respectively. Source data are available for this figure: SourceData F9.

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