Figure 1. Rtn1-GFP mislocalizes and is... | Rockefeller University Press

$Figure 1. Rtn1-GFP mislocalizes and is associated with an HSM fraction in the prp20-G282S assembly mutant. (A and B) Rtn1-GFP and dsRed-HDEL localization in the prp20-G282S mutant background was visualized by direct laser-scanning confocal microscopy. The prp20-G282S mutant (SWY3747) was grown to early log phase (23°C; A) and shifted to 34°C for 5 h (B). Single-channel fluorescence is shown from three representative cells at each temperature, and merged images are shown (right). (C) Quantitative analysis of relative Rtn1-GFP and dsRed-HDEL distribution in prp20-G282S cells at 23 and 34°C. n = 12 cells for each respective Pearson's coefficient of correlation (+1.0 = complete coincidence; −1.0 = no coincidence). Error bars represent standard deviation. (D) Biochemical fractionation was performed with rtn1-GFP GFP-nic96 prp20-G282S cells (SWY3748) grown to early log phase (23°C) and shifted to 34°C for 5 h. After lysis, subcellular fractionation was performed by differential centrifugation. Cellular equivalent fractions of the total lysate (T), pellet (P; 10,000 g), and supernatant (S; 10,000 g) fractions and 10 times the cellular equivalent of the HSM fraction (135,000-g pellet from the supernatant fraction) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with an anti-GFP. Distributions of cytoplasmic Pgk1 and nucleolar Nop1 were analyzed as controls. Bar, 2.5 µm.$

Figure 1.

Rtn1-GFP mislocalizes and is associated with an HSM fraction in the prp20-G282S assembly mutant. (A and B) Rtn1-GFP and dsRed-HDEL localization in the prp20-G282S mutant background was visualized by direct laser-scanning confocal microscopy. The prp20-G282S mutant (SWY3747) was grown to early log phase (23°C; A) and shifted to 34°C for 5 h (B). Single-channel fluorescence is shown from three representative cells at each temperature, and merged images are shown (right). (C) Quantitative analysis of relative Rtn1-GFP and dsRed-HDEL distribution in prp20-G282S cells at 23 and 34°C. n = 12 cells for each respective Pearson's coefficient of correlation (+1.0 = complete coincidence; −1.0 = no coincidence). Error bars represent standard deviation. (D) Biochemical fractionation was performed with rtn1-GFP GFP-nic96 prp20-G282S cells (SWY3748) grown to early log phase (23°C) and shifted to 34°C for 5 h. After lysis, subcellular fractionation was performed by differential centrifugation. Cellular equivalent fractions of the total lysate (T), pellet (P; 10,000 g), and supernatant (S; 10,000 g) fractions and 10 times the cellular equivalent of the HSM fraction (135,000-g pellet from the supernatant fraction) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with an anti-GFP. Distributions of cytoplasmic Pgk1 and nucleolar Nop1 were analyzed as controls. Bar, 2.5 µm.

This Feature Is Available To Subscribers Only