Figure EV4. 4D7 blocks CALR interaction with TpoR in CALR^del52 cells.

1. Western blot of TpoR immunoprecipitation under non‐reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF‐1 TpoR CALR^del52 disrupted by 8‐h treatment with 20 µg/ml 4D7 but not PBS or 20 µg/ml IgG. CALR monomers and dimers are detectable by polyclonal anti‐wild‐type CALR or anti‐mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wild‐type CALR protein; asterisk, non‐specific bands.
2. Peripheral blood mononuclear cells from PMF samples were thawed and stained for CD34, CD14, CD19 and CD3 prior to FACS sorting. Each population was collected and purity was verified prior to proceeding with any further analysis. PCR amplification of CALR exon 9 was carried out to confirm mutational status of CD34⁺ cells which were utilized in megakaryocyte differentiation assays and colony forming assays in the presence of 4D7 or IgG.
3. Representative flow cytometry plots for determination of CD41⁺/61⁺ populations from liquid culture assay from one PMF patient. Beads shown in the upper left panel with high SSC‐A. Live cell population shown in hexagon gate (top panel). CD41/61⁺ population gates shown in lower panel with % CD41/61⁺ cells indicated for unstained, IgG‐ and 4D7‐treated cells over 12 days. The number of CD41/61⁺ cells‐to‐bead ratio used to enumerate effect of 4D7 on megakaryopoiesis.

Source data are available online for this figure.