Cytokine-independent activation of STAT5 and ERK1/2 by JAK2 in UT-7/TPO CALR Del52- and Ins5-expressing cells. (A) Co-IP assay demonstrating JAK2 recruitment by c-MPL to mutant CALR. FLAG-tagged CALR Del52 or Ins5 were used as bait, and the coprecipitation of V5-tagged JAK2 in the presence or absence of c-MPL was examined. Ten percent In and the IP fraction were run as a pair. Note that the increased levels of proteins in the input when all 3 components were expressed are due to the activation of the CMV promoter in the expression vectors by the activated JAK2 pathway (Y.E., M.A., Y.H., Y.Y., S.M., Y.S., A.O., and N.K., manuscript in preparation). (B) Co-IP assay examining enhancement of c-MPL dimerization by mutant CALR proteins. V5-tagged c-MPL proteins were used as bait, and the coprecipitation of HA-tagged c-MPL was examined in the presence of CALR WT, Del52, or Ins5. (C) Immunoblot analysis of extracts (20 µg) prepared from UT-7/TPO cells infected with mock vector (vect) or UT-7/TPO cells infected with viruses expressing the indicated CALR proteins, which were cultured in the absence or presence of TPO. An arrow indicates the position of the band of the target protein based on the positive control (not shown). (D) Immunoblot analysis of extracts (20 µg) prepared from cells analyzed in (C) treated with DMSO (−) or 3 µM JAK Inhibitor I (+) for 17 hours.