Figure 2.
Investigation of Akt phosphorylation in sciatic nerves and in primary mouse Schwann cell cultures. (a) Homogenates of sciatic nerves derived from newborn MAL-overexpressing mice and wild-type littermates were analyzed by quantitative Western blot analysis. A representative blot for p75NTR, Akt, and phosphorylated Akt (Ser 473) was used for illustration. (b) The activation of Akt pathway, indicated by the ratio phospho-Akt to Akt showed no alteration in MAL-overexpressing nerves, whereas p75NTR protein expression was reduced (p = 0.08). All values were normalized to the expression of β-actin and are shown as a ratio to the respective wild-type expression. Columns represent the mean value of three independent experiments, and the error bars indicate the SD. (c) Immunofluorescence stainings on primary MAL-overexpressing and wild-type Schwann cells were performed with an antibody recognizing phosphorylated Akt (Ser 473). Cells were stimulated with 2.5 nM neuregulin1 for 15 min, and an induction of Akt phosphorylation was detected for both genotypes. DAPI as a nuclear marker was used for counterstaining. Bar: 50 µm. (d) Quantification of the mean signal intensity of phospho-Akt in unstimulated and stimulated Schwann cells revealed a significant 3.5-fold induction upon neuregulin1 treatment. Under both conditions, phosphorylation of Akt was not altered in MAL-overexpressing mice. A total of 400 pictures were analyzed per condition and genotype. Data represent the mean of three independent experiments, and error bars indicate the SEM. *p < 0.0001; NRG: neuregulin1.