A triad of zinc-binding histidine residues in CALR^del52are required for cytokine-independent growth. (A) Growth curves in Ba/F3-MPL cells expressing histidine-deficient CALR^del52 variants harboring loss of either 1 histidine (1His), 2 histidines (2His), or 3 histidines (3His). Immunoblotting (B) and intracellular phospho-flow cytometry (C) demonstrate diminished Stat3 and Stat5 phosphorylation in of Ba/F3-MPL cells expressing 2His- and 3His-CALR^del52 variants. Numbers in red indicate ratio of mean fluorescence intensity for each sample relative to isotype control. The data are representative of 2 independent experiments. (D) Immunoblotting of FLAG-immunoprecipitated proteins from 293T cells cotransfected with histidine-deficient FLAG- CALR^del52 variants and MPL-expressing vectors demonstrates abolished MPL binding capacity by 2His- and 3His-CALR^del52 variants. (E) Single-cell data for FRET intensity. FRET fluorescence units depict energy transfer between mCherry-tagged histidine-deficient CALR^del52 protein and MPL-GFP in arbitrary fluorescence units for a single cell. For each experimental condition, 100 cells were analyzed.