Fig 2: Mn-dependent enhanced a-Syn expression disturbs the phosphorylation of NMDA receptors in vivo and in vitro.a, b After treatment with Mn, western blotting was used to measure the level of a-Syn overexpression in the hippocampus. c–f The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) were measured by western blotting in vivo. n = 6. g, h HT22 cells were transfected with LV-a-Syn shRNA, and the cytotoxicity was measured using the CCK-8 assay and lactate dehydrogenase (LDH) release assay. i–m The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) and a-Syn expression were measured by western blotting in vitro. n = 4. **P < 0.01 compared to their control counterparts; # #P < 0.01, and #P < 0.05 for comparison between the Mn treatment group.

Fig 3: Effect of BMSC on NMDA receptor phosphorylation and PKC? in RVM. (a) Total proteins from RVM tissues were isolated and GluN2A (NR2A) subunit tyrosine phosphorylation was determined with anti-phosphotyrosine 1246 GluN2A (pGluN2A). At 8 weeks after primary (PRI) BMSC treatment, pGluN2A was increased in 20 P-BMSC samples (**, p < 0.01, vs. navie, n = 3). The BMSC treatment led to a return of the P-GluN2A to the naïve level (##, p < 0.01, vs. 20 P/8 weeks). There was a trend for increase in pGluN2A at 1 week after BMSC treatment, which was likely an effect of injury. (b) The levels of PKC? were reduced in PRI BMSC-treated rats at the 8-week time point (#, p < 0.05, vs. 20 P/8 weeks). ß-actin was a loading control. N = 3/group, ANOVA with post-hoc comparisons. (c) Localization of PKC?- and GluN2A-like immunoreactivity in RVM neurons. Brainstem tissue sections were processed for PKC? and GluN2A double immunofluorescence staining. PKC? immunoreactivity (red, Cy3) is colocalized with NeuN (green, Cy2). Overlap of images of the left and middle columns reveals double fluorescence (right, yellow/orange). Arrows indicate examples of double-labeled neurons. Scale = 0.25 µm.