Binding of mutant calreticulin to MPL is required to transform hematopoietic cells but binding alone is insufficient for cytokine-independent growth. (A) Schema depicting serial C-terminal truncation mutants of mutant CALR. (B) Immunoblotting of FLAG-immunoprecipitated proteins and whole cell lysates from 293T cells cotransfected with wild-type FLAG-CALR (CALR^WT), FLAG-CALR 52-bp deletion (CALR^MUT), or FLAG-CALR 52-bp deletion serial C-terminal truncation mutants (CALR^MUT Δ10-Δ36) demonstrates that mutant CALR truncated up to Δ36 still binds to MPL. (C) Immunoblotting demonstrates phosphorylation of Stat5 and Stat3 in Ba/F3-MPL cells expressing CALR^MUT and truncation variants Δ10, Δ18, and Δ28, but not Δ36. (D) Immunoblotting demonstrates phosphorylation of MPL in Ba/F3-MPL cells expressing CALR^MUT and Δ28, but not Δ36. (E) Growth curves in Ba/F3-MPL cells expressing CALR^WT, CALR^MUT, or CALR^MUT C-terminal truncation variants demonstrates that only severe truncation of the mutant CALR C terminus (Δ36) abolishes the transforming capacity of mutant CALR. (F) Immunoblotting of FLAG immunoprecipitated proteins from 293T cells co-transfected with FLAG-CALR 52 bp deletion (CALR^MUT) and glutathione S-transferase (GST)–tagged full-length MPL, GST-tagged MPL intracellular + transmembrane domains, or GST-tagged MPL extracellular + transmembrane domains demonstrates that mutant CALR binds to the extracellular domain of MPL. (G) Immunoblotting of FLAG immunoprecipitated proteins from 293T cells coexpressing FLAG-tagged mutant CALR and MPL YF variants demonstrates that mutations of intracellular tyrosine residues on MPL does not affect the ability of mutant CALR to bind to MPL. (H) Immunoblotting demonstrates that phosphorylation of Stat5 is abrogated in Ba/F3 cells expressing MPL YF variants harboring loss of Y626. (I) Growth curves in Ba/F3 cells stably expressing MPL-YF variants demonstrate that all 3 intracellular tyrosines play a role in supporting cytokine independent growth in Ba/F3 cells mediated by mutant CALR. WB, western blot.