Transfection and western blot analysis

For the transfection, 1 μg of plasmid DNA was suspended in 100 μL of PBS. Then, 4 μL of PEI (1 mg/mL) was added to the solution and gently mixed. This solution was incubated at room temperature for 20 min and then mixed with 293TT cells in a 12-well plate. Twenty-four hours after transfection, the cells were washed once with PBS and then mixed with 1 × SDS loading buffer and incubated at 95 °C for 10 min. In some experiments, 10 μM MG132 was added to the medium 12 h before cell harvest.

The cell lysate was loaded onto a SuperSep Ace 5–20% SDS PAGE (Wako, Tokyo, Japan) or a 10% SDS PAGE and electrophoresed in running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS) for 50 min at 180 V. The separated proteins were transferred to a nitrocellulose membrane using a Atto Western Blotting System (Tokyo, Japan) with EZ Fast Blot (Atto) for 15 min at 0.25 A per mini gel. The membrane was blocked with 5% skim milk at 37 °C for 20 min, and then washed five times with wash buffer (PBS with 0.5% Tween 20) at room temperature for 5 min with shaking. The membrane was incubated with primary antibody (1:3,000 dilution in PBS with 0.1% BSA) at 37 °C for 15 min. After washing with wash buffer at room temperature (five times for 5 min each), the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP 1:3,000 dilution in 5% skim milk) at 37 °C for 15 min. After washing five times with the wash buffer at room temperature (5 min each), the protein was detected with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific), and analyzed by LAS-3000 with MultiGauge software Version 2.2 (Fujifilm, Tokyo, Japan, https://www.ualberta.ca/biological-sciences/media-library/mbsu/fla-5000/mulitgauge20.pdf).