Anti kar2

To analyze total ubiquitin conjugates, whole-cell extracts were prepared from exponential-phase cultures. Cells were normalized by optical density; cell pellets were resuspended in 1× Laemmli loading buffer (LLB) and boiled for 5 min. For all other immunoblots, extracts were prepared by a lithium acetate/sodium hydroxide method as follows. Exponential-phase cultures were normalized by optical density, treated with 2 M lithium acetate on ice for 5 min, and then treated with 0.4 M sodium hydroxide on ice for 5 min. Cell pellets were resuspended in 1× LLB and boiled for 5 min. Analysis was by standard SDS–PAGE followed by immunoblotting. For cycloheximide chase analyses, cycloheximide (100 µg/ml) was added at time 0 to inhibit protein synthesis, and extracts were prepared as described at the indicated time points. Note that the experiments of Figure 3, A–C and E, were carried out at 37°C. Where indicated, the unfolded protein response was induced by treating cells with tunicamycin at 5 μg/ml for 1 h.
The following antibodies were used: anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Pgk1 (459250; Invitrogen), anti–HA-peroxidase (12013819001; Roche), anti-actin (MA511866; ThermoFisher), anti–phospho-eIF2α (9721S; Cell Signaling Technology), and anti-Kar2 (sc-33630; Santa Cruz Biotechnology).