Cell surface localization of mutant CALR with no paracrine activation for UT-7/TPO cell growth. (A) Cell surface proteins were biotinylated, purified, and analyzed by immunoblot for the detection of FLAG-tagged CALR WT and Ins5, and c-MPL. The relative intensity of each band is indicated. Total cell lysate corresponding to 10% of cells used for surface protein isolation were run as “total.” Note that the expression of CALR WT is significantly higher than Ins5, as shown in Figure 1B. Representative data from multiple experiments are presented. (B) Subcellular distribution of CALR WT, Ins5, and c-MPL were analyzed. cyto, cytosol; mem, membranes; nuc, nuclear; Csk/Dbr, cytoskeleton and debris; total, total cell lysate prepared from same number of cells used for fractionation. Note that for the detection of FLAG-tagged CALR WT, samples analyzed were diluted 8-fold because of elevated protein expression (Figure 1B). Markers for each fraction such as GAPDH (cytosol), Calnexin (ER), Na+/K+ ATPase (extracellular membrane), and Lamin B (nuclear) were analyzed. Representative data from multiple experiments are presented. (C) Cell count analysis for tracking dye labeled UT-7/TPO cells. UT-7/TPO cells were cultured with (upper) or without (lower) UT-7/TPO CALR Ins5 in the absence (left) or presence (right). Note that when starting the culture, 2 times more cells were cultured in UT-7/TPO alone (bottom) to equalize the starting cell number to cocultured cells (top).