Figure 2.

Aβ₄₂ activates Src-kinase in nanomolar concentrations. (A) Co-localization studies with Proximity Ligation Assay. Close proximity of Na,K-ATPase α1-subunit and Src kinase in the SH-SY5Y neuroblastoma cells. The confocal merged image of Hoechst fluorescence (blue), RNASelect (green) and Duolink Red (red) fluorescence is presented. Scale bar—50 µm. (B) The effect of Aβ₄₂ on the Na,K-ATPase transport activity in SH-SY5Y cells. K⁺ (Rb⁺) influx after 30 min treatment with 100 nM Aβ₄₂. Total Rb⁺ influx into the cells was measured in the absence of ouabain (Total); “Passive” denotes ouabain-resistant component of Rb⁺ influx in the sample where ouabain was added. Difference between the total and the passive fluxes gives the active (Active) Rb⁺ influx mediated by the Na,K-ATPase. (C) The changes in Na,K-ATPase levels in SH-SY5Y neuroblastoma cells after 30 or 60 min of incubation with 100 nM Aβ₄₂. Na,K-ATPase levels were evaluated by flow cytometry. (D,E) Dose-dependent activation of Src by Aβ₄₂. The ratio of phospho(Tyr)-416 Src to the total Src has been calculated. The phosphorylated and total Src levels have been measured with Western blot in SH-SY5Y neuroblastoma cells treated with 100 nM, 500 nM and 2 µM of Aβ₄₂ for 30 min and normalized for control. Mean values ± SD from at least three independent experiments are shown. *—p < 0.05, ***—p < 0.001 compared to the control, ns—nonsignificant.