Ribosome Ubiquitination During ER Stress

To assess ribosome ubiquitination during ER stress, ribosomes were purified with Myc-tagged ubiquitin (Myc-Ubi) and the FLAG-tagged ribosomal protein uL23 (uL23-FLAG), as described previously^{22 (link)}. Yeast cells harbouring pCUP1p-MYC-UBI and pRPL25(uL23)-FLAG were cultured in 800 mL of synthetic complete medium. To induce the expression of Myc-Ubi, the cells were cultured in the presence of 0.1 mM Cu²⁺ for 1 h, followed by the addition of Tm to a concentration of 1 µg/mL and harvesting at the indicated time points. Cell lysates were prepared, and FLAG-tagged ribosomes were purified using M2 FLAG-affinity resin (Sigma), as described previously^{22 (link)}. Affinity purified samples were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or western blotting with an anti-Myc antibody.

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Matsuki Y., Matsuo Y., Nakano Y., Iwasaki S., Yoko H., Udagawa T., Li S., Saeki Y., Yoshihisa T., Tanaka K., Ingolia N.T, & Inada T. (2020). Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome. Scientific Reports, 10, 19669.

Publication 2020

M2 FLAG-affinity resin

Anti antibody Cell Coomassie brilliant blue Cultured medium Resin Ribosomal protein Ribosomes Sds page Ubiquitin Ubiquitination Yeast

Corresponding Organization :

Other organizations : Tohoku University, University of California, Berkeley, Tokyo Metropolitan Institute of Medical Science, University of Hyogo

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Variable analysis

independent variables

Induction of Myc-Ubi expression with 0.1 mM Cu2+ for 1 h
Addition of Tm to a concentration of 1 µg/mL

dependent variables

Ribosome ubiquitination during ER stress

control variables

Yeast cells harboring pCUP1p-MYC-UBI and pRPL25(uL23)-FLAG
Culturing in 800 mL of synthetic complete medium
Purification of FLAG-tagged ribosomes using M2 FLAG-affinity resin

controls

Positive control: Not specified
Negative control: Not specified

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