Fig 1: Neutrophil polarization is not caused by activation of the known IGF1 pathways (A) Immunoprecipitation showed the presence of the insulin-like growth factor 1 receptor (IGF1R) and insulin receptor (InsR) in bone marrow derived neutrophils (B) 10 min IGF1 and insulin treatment caused phosphorylation of ERK (pERK) as shown by western blot. Data is normalized to pERK in untreated neutrophils. (C) Western blot analysis showed the presence of AKT in bone marrow derived neutrophils, however none of our treatments caused AKT phosphorylation (pAKT) after 10 min treatment. As a positive control AKT phosphorylation of 3T3 cells after insulin treatment is shown on the same western blot. (D) Akt1 and 2 expression in Tie2-Cre Akt1fl/fl and Tie2-Cre Akt2fl/fl mice was assessed by Western blot. (E-F) N1 and N2 polarization after our treatments was unaffected in Tie2-Cre Akt1fl/fl (E) and Tie2-Cre Akt2fl/fl (F) neutrophils as shown by normal upregulation in quantitative RT-PCR of Nos2 after LPS/IFNγ treatment and Arg1 and Retnla after IL4 and IGF1 treatment. LPS: Lipopolysaccharide, IFNγ: interferon-gamma, IL4: Interleukin 4, IGF1: Insulin-like growth factor 1, Nos2: nitric oxide synthase 2, Arg1: Arginase 1, Retnla: resistin like alpha. X0 values normalized to Nudc (nudC nuclear distribution protein) expression of single measurements are shown. Bars represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001.