Supplementary Figure 3: Stau1 is associated with actively translating ribosomes.
From: Staufen1 senses overall transcript secondary structure to regulate translation
a, Sucrose sedimentation analysis of Stau1 expressing cells. Cells were incubated with cycloheximide (100 μg.ml-1) for 10 minutes before cell lysis. Obtained lysates were treated with RNase A or not before loading them on top of a 10-50% linear sucrose gradient and subjected to ultracentrifugation. After fractionation, obtained samples are analyzed by western-blotting using anti-Stau1 antibodies. b, Ribosome run-off assay for Stau1 expressing cells. Cells were incubated with cycloheximide for 10 minutes (upper panel) or harringtonine (2 μg.ml-1) for 10 minutes (middle panel) or 40 minutes (lower panel) before adding cycloheximide and further incubating cells for 10 minutes before lysis. Cells lysates are then loaded on top of a 10-50% linear sucrose gradient and subjected to ultracentrifugation. After fractionation, obtained samples are analyzed by western-blotting using anti-Stau1 and anti-RPL26 antibodies.
