The interactome of CALM-AF10 FP in AML. (A) Schematic representation of the CALM-AF10-flag pulldown and MS. Primary AML cells with a 3xFlag-tagged CALM-AF10 FP and an IRES-linked tdTomato fluorescent marker were used for cell lysis and conjugated with Flag-M2 beads overnight. Following trypsin digestion, MS analysis was performed (see “Methods” for more details). Similar studies were done on MLL-AF10-flag AML cells. (B) Log2 fold-change (x-axis) and adjusted P values (y-axis) of proteins differentially identified in 3xFlag-CALM-AF10 IP samples compared with IgG are plotted. Murine CALM-AF10 AML cells were used for this study. (C) IP using anti-Flag antibodies in CALM-AF10 or MLL-AF10 AML cells blotted with an antibody for endogenous Jak1. β-actin is used as a loading control. (D) Immunoblotting for the endogenous Jak1 protein in murine iCALM-AF10 or iMLL-AF10 AML cells in the FP-On compared with FP-Off cells is shown together with Jak1 levels in MG132-treated AF10 FP-Off cells. β-actin is used as a loading control.