Ubiquitination assay

HEK293T cells were transiently transfected with 3 μg pcDNA5 vector expressing CALR WT and CALR del52 together with or without 4 μg pcDNA3-ubi-HA vector using TransIT-LT1 reagent (Mirus, Madison, USA). After 24 h, the medium was discarded and replaced by fresh DMEM containing 10 μM MG132 for 20 h, and cell lysates were generated. For the immune precipitation (IP) assay, 40 μl Protein G Sepharose (GE Healthcare, Freiburg, Germany) was mixed with 2 μg anti-flag antibody for 4 h at 4 °C and subsequently incubated with 1 mg lysate in 1 ml RIPA buffer overnight, rocking at 4 °C with three times washing using cold PBS in between. The sepharose was then washed three times with RIPA buffer and resuspended in Laemmli/RIPA solution. After denaturation, bound proteins were separated and prepared for Western blotting.