Fig. 4

Degradation of instable CALR mutants is proteasome-independent and mutants get stabilized by MPL expression. a The expression of indicated CALR-flag constructs in 32D cells +/− ectopic MPL was confirmed by RT-qPCR. The expression is depicted as percentage to Gapdh. Experiments were performed in triplicates. Mean and SD are indicated. **P < 0.01, ***P < 0.001. b 32D empty vector (EV), WT CALR-positive, or del52-positive cells were treated with 10 μM MG132 for indicated periods of time. Afterwards, cells were starved in WEHI-free medium for 4 h and lysates were prepared for SDS-PAGE and Western blotting. Indicated antibodies were used for immunodetection to show protein stability. c HEK293T cells were transfected with HA-tagged ubiquitin and the indicated CALR-flag constructs. After 24 h, 10 μM MG132 was added for 20 h and protein lysates were prepared. Immunoprecipitation (IP) was performed with flag antibody followed by SDS-PAGE and Western blotting. In addition, whole cell lysates were used for Western blotting. d Autophagosomal inhibition was performed in 32D EV, WT CALR, del52, and ins5 expressing cells. The 32D cell lines were treated with the inhibitor spautin-1 for the indicated time. Lysates were prepared, and SDS-PAGE and Western blotting were performed. Antibodies detecting mutated CALR (CALR mut), CALR, LC3I-II, and GAPDH were used for immunostaining. LC3I-II served as control for successful autophagosomal inhibition. e 32D cells expressing EV, WT CALR, del52, or ins5 mutant +/− MPL receptor were starved overnight. Lysates were prepared and subjected to SDS-PAGE and Western blotting. The PVDF membrane was subjected to the indicated antibodies