Zinc binding by CALR^del52is required for homomultimerization and enables MPL activation. (A) Schematic depicting co-immunoprecipitation assay for detecting FLAG-tagged and V5-tagged CALR^del52 multimers. (B) FLAG-pulldown assays demonstrating CALR^del52 lectin variants retain ability to bind to V5-CALR^del52. (C) FLAG-pulldown assays demonstrating 2His- and 3His-CALR^del52 are compromised in ability to bind V5-CALR^del52 compared with 1His-CALR^del52. (D) FLAG-pulldown assays of cells transfected with FLAG- and V5-tagged CALR^WT (lane 1), CALR^del52 (lane 2), CALR^del52 D135A lectin variant (lane 3), or 3His-CALR^del52 variant (lane 4) demonstrating that CALR^del52 and CALR^del52 D135A are capable of homomultimerization, but not CALR^WT and 3His-CALR^del52. (E) Affinity chromatography demonstrating 2His- and 3His-CALR^del52 bind less efficiently to zinc resin compared with 1His-CALR^del52. (F) Schematic depicting co-immunoprecipitation assay for detecting heteromeric complexes comprising FLAG-tagged and V5-tagged CALR^del52 proteins and MPL. (G) FLAG-pulldown assays demonstrating zinc chelator TPEN treatment disrupts CALR^del52 multimerization and MPL binding in 293T cells.