STAT3 is phosphorylated on S727, not Y705, in K-RasG12D-driven MPNs in vivo. STAT3-Flox mice of the following genotypes, Mx:STAT3F/+ (M:F/+), Mx:K-Ras^G12DLSL:F/+ (M:K:F/+), or K-Ras^G12DLSL:STAT3F/+ (K:F/+), were injected with a single 100-µg dose of poly(I:C). Mice were allowed to recover for 2 weeks before peripheral blood was collected, red blood cells lysed, and total leukocytes assessed for STAT3 phosphorylation on S727 (pS STAT3) or Y705 (pY STAT3) by immunoblotting (A). Antibody against total STAT3 served as a loading control. As a positive control for STAT3 phosphorylation, wild-type mice were injected with 30 µg of LPS 4 hours prior to harvesting peripheral blood leukocytes (LPS). (B) After 2 weeks of recovery following poly(I:C) injection, bone marrow was harvested from Mx:F/+ (Control) or Mx:K-Ras:F/+ mice (M:K) and expression of (i) pS727 STAT3, (ii) pY705 STAT3, (iv) pSTAT5, and (v) pERK was determined in lineage-negative, c-Kit⁺ cell populations by flow cytometery using phospho-specific antibodies. As a positive control for STAT3 Y705 phosphorylation (iii), mice were injected with 30 µg of LPS 4 hours prior to harvesting bone marrow. Pooled data from 3 animals for each genotype are plotted in the histogram showing the MFI ± standard deviation. Statistical significance was calculated using the Student t test. *P < .05, ***P < .001. MFI, mean fluorescence intensity.