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. 2021 Apr 5;5(7):1922–1932. doi: 10.1182/bloodadvances.2020002402

Figure 2.

Figure 2.

A triad of zinc-binding histidine residues in CALRdel52are required for cytokine-independent growth. (A) Growth curves in Ba/F3-MPL cells expressing histidine-deficient CALRdel52 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-CALRdel52 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- CALRdel52 variants and MPL-expressing vectors demonstrates abolished MPL binding capacity by 2His- and 3His-CALRdel52 variants. (E) Single-cell data for FRET intensity. FRET fluorescence units depict energy transfer between mCherry-tagged histidine-deficient CALRdel52 protein and MPL-GFP in arbitrary fluorescence units for a single cell. For each experimental condition, 100 cells were analyzed.