Absence of functional CALR leads to MPO deficiency through proteasomal degradation of MPO. (A) Expression of hMPO protein tagged with an Emerald-tag in mouse embryonic fibroblasts (K41) and mouse embryonic fibroblasts derived from a CALR knockout mouse (K42). A proteasome inhibitor (MG132) and a lysosome inhibitor (Bafilomycin) were added to the cultures as indicated. MPO protein levels were assessed by western blot. The expected size for apopro-MPO (with Emerald-tag), and β-Actin are 130 kDa and 42 kDa, respectively. The asterisk indicates an unspecific band also observed in untransfected K41 and K42 cells. (B) RFI of Emerald assessed by flow cytometry in the same conditions as in (A). The experiment was repeated 3 times. (C) Relative expression of hMPO mRNA to that of mouse glyceraldehyde-3-phosphate dehydrogenase in K41 and K42 cells transfected with hMPO. Results are expressed as mean ± standard deviation. *P < .05; **P < .01 (unpaired Student t test).