Fig 2: Quantification of HGF-induced PI3K/AKT signaling at the single cell level. (A) Confocal image of an individual mCherry-AKT transfected primary mouse hepatocyte is shown as overlay of the Hoechst, WGA-Alexa488, and mCherry-AKT signal with the signals from different channels in artificial-coloring. The graphical representation shows the tracked membrane signal in blue and the rim of the non-membrane cytoplasmic region in yellow with the localization of the two nuclei in the center. (B) Magnification of a subselection showing to the left of the tracked membrane section that is marked in blue the intracellular cytoplasmic space whereas to the right the bright green signal due to background staining of the WGA-Alexa488 visualizes the extracellular space. In the lower panel the quantification areas for mCherry-AKT intensity derived by the membrane tracking are depicted as blue (membrane associated) and yellow regions (intracellular reference area). (C) Signals from 25 individual single cell traces in response to 40 ng/ml HGF stimulation are represented in different colors. (D) Average of single cell traces are depicted for untreated controls (n = 12) in blue, after stimulation with 40 ng/ml HGF (n = 50) in red, and for cells pretreated with LY294002 for 30 min prior to HGF stimulation (n = 15) in green. Error bars represent the standard error of the mean.

Fig 3: Representation of experimental and model-derived CVs. The average of the kinetics of mCherry-AKT membrane association in 10 individual cells for (A) the clone Hepa1_6-D8 and (B) clone Hepa1_6-E2 are shown with the standard error of the mean indicated for each time point. (C + D) The calculated dynamics for 10 simulated cells with extrinsic noise contribution as given by the parameters are shown. For the clones (E) Hepa1_6 D8 and (F) E2 the CV's for the experimental fluctuations in mCherry-pAKT (blue line), theoretical intrinsic fluctuations in mCherry-pAKT (green line), and the corresponding combination of extrinsic and intrinsic fluctuation (red line) are plotted.

Fig 4: PKC? and PKM? protein localize to central neurons in the dorsal horn and are increased by BDNF treatment. (A) Slices were prepared from mice and stained with an atypical PKC (aPKC) antibody (red) and the neuronal marker (found in soma and post-synaptic sites) SAP-102 (green). The top panel shows that aPKC immunoreactivity is localized primarily to the spinal dorsal horn. Lower panels (from inset) show that aPKC immunoreactivity is localized almost exclusively to neurons in the dorsal horn. (B) qPCR was used to assess mRNA expression of aPKC isoforms in spinal cord and kidney (N = 3). Spinal SNSs were prepared and mRNA or protein was isolated. Spinal SNSs were enriched for GluN1 mRNA and were ßIII tubulin poor, they were likewise enriched in PSD95 protein as shown by Western blotting compared to equal protein concentration of whole spinal cord homogenate (input). C) SNSs were isolated from mouse lumbar spinal cord and exposed to increasing concentrations of BDNF for 15 min. BDNF dose-dependently increased mTOR, AKT and p70S6 kinase (p70) phosphorylation when standardized to total protein levels. BDNF also increased total levels of PKC? PKM? and CaMKIIa when compared to loading control ßIII tubulin (N = 6). * p < 0.05, *** p < 0.001, one way ANOVA with Bonferroni post hoc test compared to Veh.

Fig 5: eNOS/NO modulates VE-cadherin Y685 phosphorylation via c-Src in a VEGFA/VEGFR2-dependent manner.VEGFA through VEGFR2 and its phosphosite Y1212 induces a chain of consecutive reactions in endothelial cells: phosphorylation of AKT at S473 and eNOS at S1176. The VEGFR2 phosphosite Y949 mediates phosphorylation of c-Src at Y418 and of VE-cadherin at Y685. Combined, these activating phosphorylation reactions disrupt the vascular barrier by dissociating VE-cadherin’s homophilic interactions, resulting in macromolecular leakage. eNOS/NO regulates activation of c-Src to enhance VE-cadherin Y685 phosphorylation and internalization.