sTFR1 is a carrier protein for mutant CALR. (A) Allele burden and mutation profile of patients included in this study the analysis. (B) Identification of partners of plasmatic mutant CALR. Plasma from 5 patients with mutated CALR and 5 healthy controls was isolated by IP with biotinylated antimutant CALR antibody. Immunoblot shows the presence of mutant CALR in the IP product detected with a different antimutant CALR antibody. The IP product was analyzed by nontargeted MS to identify interacting partners. (C) Truncated violin plot of relative sTFR1 amount found coimmunoprecipitating with plasma CALR-del52. Negative control corresponds to the IP product after anti–mutant CALR IP to control for nonspecific binding to anti–mutant CALR antibody. (D) Stability study of rhCALR-del52 in medium with 10% fetal bovine serum (FBS), with or without addition of 2 or 4 μg/mL of rhTFRC. rhCALR-del52 mutant in different media was maintained at 37°C for various lengths of time and measured by ELISA before analysis using a 1-phase decay model (Prism6) to determine the averaged half-life. Values represent mean of triplicate ± SD. (E) Representative confocal microscopy pictures of HEK293T cotransfected with either CALR WT or CALR-del52 fused to green fluorescent protein at the C-terminus and TFR1 fused to mCherry at its C-terminus. Scale bars represent 10 μm. Microscopy analysis shows colocalization between CALR-del52 and TFR1 in subcellular compartments with high correlation between the 2 constructs. (F) IP and glycosylation profile analysis of endogenous TFR1 from UT-7/Tpo or UT-7/Tpo CRISPR CALR-del52. Western blot shows the TFR1 forms analyzed by MS. Data represent the percentage of peptide-spectrum match of immature N-glycans (high mannose) present on residue Asn251 in the cleaved or full form of TFR1.