Extended Data Figure 10: Stability of Gis2–HA and Nog2–HA.
From: A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death
a, b, Relative steady-state levels of Gis2–HA and Nog2–HA in proteasomal mutants. Data are mean ± s.d. of 3 and 5 independent experiments for Gis2–HA and Nog2–HA, respectively (*P < 0.05; **P < 0.01; unpaired Student’s t-test). c, d, Half-life of Gis2–HA and Nog2–HA in the wild-type and AAC2A128P (A128P) cells. Data are mean ± s.d. of three experiments (P < 0.05 for Gis2–HA and P = 0.15 for Nog2–HA, unpaired Student’s t-test). e, Western blot analysis showing no evidence of Gis2–HA ubiquitination in the poc4Δ, ump1Δ and rpn4Δ cells. The ump1Δ and rpn4Δ cells are temperature-sensitive because of defective proteasomal function. Cells were grown at the non-permissive temperature (37 °C) before being lysed for protein extraction and SDS–PAGE. f, Gis2–HA was immunoprecipitated from the wild-type (WT) and AAC2A128P (A128P) cells and analysed by western blot using antibodies against HA (left) and ubiquitin (right). Note that the immunoprecipitation-purified full-length Gis2–HA migrates slower than the protein in the cell lysate, probably due to posttranslational modification during immunoprecipitation. The cryptic modification is unrelated to ubiquitination, based on the lack of reactivity with the anti-ubiquitin antibody.
