Preferential binding of c-MPL and mutant CALR proteins. (A) Co-IP assay for the detection of endogenous c-MPL and retrovirally expressed FLAG-CALR proteins in UT-7/TPO cells. Note that the FLAG-tagged CALR Del52 band overlaps with the heavy chain from IgG (*) used for immunoprecipitation. The overlapping bands for Del52 and IgG were not significantly more intense than that of IgG alone because of a lower accumulation of Del52, as shown in Figure 1B. Note that c-MPL accumulated at a higher level in CALR Del52- or Ins5-expressing cells compared with CALR WT- or vector control-expressing cells partly, if not all, owing to an increased mRNA level (data not shown). Ten percent input (In) and the IP fraction were run as a pair in panels A-C. c-MPL appears as a doublet in the blot. (B-C) Co-IP assay for the detection of coexpressed c-MPL and CALR proteins in HEK93T cells. FLAG-tagged CALR (B) or V5-tagged c-MPL (C) was used as bait.