Fig. 7.

Western blot analysis. To validate the specificity of the antibodies, western blot analysis was performed with the two best antibodies against pAkt (#4060 and #2965) and pMAPK (#4370 and 9101) on lysate from HEK 293 cells, which had been stimulated with insulin (10 nM) and lysate from hippocampal neurons, which had been stimulated with BDNF (1 nM). Clear bands with the correct size for both pAkt (A–C, upper panel) and pMAPK (D–F upper panel) were detected with the used antibodies. The antibodies were able to detect a significant increase of the phosphorylated proteins after stimulation, observed as strong increases in band intensity between unstimulated (−) samples and stimulated (+) samples. To ensure that the increases in band intensities after stimulation were not due to different levels of Akt and MAPK, the membranes were stripped and visualized for total amount of Akt (A–C, lower panel) and MAPK (E–F, lower panel), showing that the observed increases in pAkt and pMAKP were indeed due to insulin/BDNF stimulation. The lanes to the left in figure D–F are molecular weight makers.