Traffic through the secretory pathway is required for TpoR activation by CALR del52. (A) CALR del52 mutant lacking the signal peptide does not activate STAT5 transcriptional activity in γ2A cells via TpoR. (B) Blocking the anterograde secretion pathway by BFA treatment of 16 hours (started 12 hours after transfection) leads to a complete inhibition of STAT5 transcriptional activity via WT TpoR in transfected γ2A cells. (C) Confocal analysis confirmed that 16 hours of BFA treatment of mouse embryo fibroblasts knock out for Calr and stably transduced with CALR variants and TpoR redistributes CALR del52 to the ER. (D) BFA treatment 12 hours posttransfection (for 16 hours) does not block activation of TpoR LL-AA by CALR del52 or Tpo, as assessed by STAT5 transcriptional activity in transfected γ2A cells. (E) Treatment of Ba/F3 TpoR eCRISPR/CALRdel52 with Dynasore hydrate (50 μM), which blocks endocytosis, increases STAT5 transcriptional activity. Shown are averages of 3 independent experiments each with 2 to 3 biological replicates. Statistical analysis (jmp pro12) was performed by using the nonparametric multiple comparisons Steel test with a control group (A-B) or by the unpaired nonparametric 2-tailed Student t test (E).