Figure EV1. Characterization of 4D7 antibody as a potential therapeutic tool.

1. Western blot screening of various mutant CALR antibody clones produced from hybridomas in TF‐1 TpoR and TF‐1 TpoR CALR^del61 cells. Varying levels of intensity can be observed compared to commercial Dianova monoclonal mutCALR antibody. 4D7 is able to detect mutant CALR protein in CALR^del61 cells but not in CALR wild‐type TF‐1 cells.
2. Biological replicate Scatchard analyses of 4D7 using ¹²⁵I‐labelled and unlabelled 4D7 bound to full‐length peptide (n = 3 technical replicates).
3. TF‐1 cells expressing TpoR and CALR^del61 or CALR^del52 demonstrate factor independence in absence of TPO. Cells were cultured in the presence or absence of 10 ng/ml TPO (n = 3 biological replicates).
4. Paracrine CALR‐mutant protein is not sufficient to maintain TPO‐sensitive cells in culture. TF‐1 TpoR and TF‐1 TpoR CALR^del61 cells were seeded at the same density and cultured in the presence or absence of TPO. Cells were separated by semi‐permeable membrane in a horizontal co‐culture system. Cell populations on either side of the membrane were counted every 24 h over 4 days in triplicate and representative images were taken on day 4. Exogenous CALR secreted by TF‐1 TpoR CALR^del61 was unable to assist growth of factor‐dependent TF‐1 TpoR cells. Scale bar indicates 100 µm (n = 3 biological replicates).
5. Histogram overlays showing fluorescence intensity of unstained and PE‐conjugated IgG2a isotype control in TF‐1, TF‐1 TpoR, TF‐1 TpoR CALR^WT and TF‐1 TpoR CALR^del61 compared to 4D7 conjugated to PE.

Source data are available online for this figure.