Figure 5

Human ASPL partially suppresses some defects of a yeast ubx4Δ mutant. (a) Serial dilutions of wild type or ubx4 Δ strains transformed with either a control plasmid (Mock) or an ASPL expression vector (ASPL) were spotted onto solid media with or without 0.2 μg/mL cycloheximide (CHX). The plates were incubated at 30°C until colonies formed. (b) Extracts from the indicated strains were resolved by SDS-PAGE and blotting using antibodies to ASPL and, as a loading control, the constitutive Pma1. (c) Wild type (wt) and ubx4 Δ cells expressing CPY* were treated with cycloheximide. At the indicated times the amount of CPY* was analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (d) Wild-type yeast and cdc48-3 P _GAL -3HA-UBX4 mutant expressing Pre6-GFP and nuclear envelope marker Nup49-mCherry were grown in synthetic medium containing glucose and analyzed by fluorescence microscopy. (e) Yeast 2 μ plasmids carrying ASPL or UBX4 genes were transformed into PRE6-GFP cdc48-3 P _GAL -3HA-UBX4 strain. Transformation with the empty vector pRS426 serves as negative control. Cells were grown in synthetic medium containing galactose or glucose to induce or repress, respectively, UBX4 and analyzed by fluorescence microscopy. (f) The percentage of cells containing Pre6-GFP foci were quantified and shown as average and standard deviation for three independent experiments. Bar, 2 μm. (**, p < 0.01).