Fig. 2

32D cells expressing CALR mutants show increased mRNA and protein levels of the megakaryocytic transcription factor NF-E2. a Detection of Nfe2 mRNA expression by RT-qPCR in the indicated 32D cells. The experiments were performed in triplicates. SD is indicated. *P < 0.05, **P < 0.01, ***P < 0.001. b Lysates were prepared of 32D cells expressing WT CALR, del52, and ins5 mutant as well as of outgrown 32D del52 cells, and NF-E2 protein was detected in Western blotting. In b and d, GAPDH served as loading control and was used for the calculation of NF-E2 expression ratios. c HL60e EV (empty vector), CALR WT, CALR del52 and CALR ins5 cells were used to prepare lysates, and SDS-PAGE and Western blotting were performed. Indicated antibodies have been used for immunostaining. CALR mut antibody showed unspecific binding to ectopic WT CALR. d HL60e cells expressing empty vector (EV), WT CALR, del52, or ins5 were analyzed for NF-E2 expression, and expression ratios were calculated as in b