Proteasome activity assay

In vitro proteasome activity assay was carried out using the 20S Proteasome Activity Assay kit (Sigma-Aldrich catalog # APT280) and following the manufacturer protocol. Briefly, media was aspirated from wells (~600 000 cells/well) and replaced with lysis buffer. Cells were scraped and lysed, and sample protein concentration was quantified and normalized using the Pierce BCA Protein Assay Kit as described above for Immunoblotting. Sample lysates of equal protein amount were added to an assay mixture of proteasome substrate (Suc-LLVY-AMC), assay buffer and water (adding up to 100 ul) and then incubated for 2 h at 37 C in a 96-well plate. Fluorescence intensity was read using a 380/460 nm filter set on FLUOstar Optima microplate reader (BMG Labtech).