Fig 1: XMD8-92 inhibited the activity of the ERK5 signaling pathway. The protein expression of MEK5 and NUR77 was detected by western blot after the MC3T3-E1 cells were pretreated with 10 μM XMD8-92 for 1 h followed by 100 nM liraglutide treatment for 48 h (a). Quantitative analysis of the protein level of MEK5 (b) and NUR77 (c) in MC3T3-E1 cells after being pretreated with 10 μM XMD8-92 for 1 h followed by 100 nM liraglutide treatment for 48 h, aP < 0.05 compared with control, bP < 0.05 compared with 100 nM liraglutide, and cP < 0.05 compared with XMD8-92 group. C: control group; LIR: 100 nM liraglutide; X: XMD8-92, X + LIR: XMD8-92 with 100 nM liraglutide.
Fig 2: Liraglutide-activated ERK5 signaling pathway protein expression. MC3T3-E1 cells were treated with different concentrations of liraglutide for 48 h, and then the protein expression of MEK5, NUR77, ERK5, and p-ERK5 was measured by western blot (a, d). Quantitative analysis of the protein level of MEK5 (b), NUR77 (c), and p-ERK5 (e) in MC3T3-E1 cells after treatment with liraglutide for 48 h. aP < 0.01 compared with 0 nM, bP < 0.01 compared with 10 nM, and cP < 0.01 compared with 100 nM.
Fig 3: Mechanistic diagram. HG exacerbated hippocampal injury following TBI, largely by disrupting the balance between pro- and anti-inflammatory factors and inducing apoptosis in hippocampal neurons. The MEK5/ERK5 pathway was found to participate in both of these processes.
Fig 4: HG reduced cell viability and aggravated neuroinflammation by inhibiting the MEK5/ERK5 pathway. (A) Cellular viability was detected using a CCK-8 assay. The transfection efficiency was confirmed using (B) Western blot analysis and (C) qRT-PCR. **P < 0.01 vs. the pcDNA3.1 vector group. Bar graphs illustrate the relative mRNA levels of (D) IL-1ß, (E) TNF-a, (F) IL-10 and (G) TGF-ß. ß-actin was used as the qRT-PCR control. Data are presented as the mean ± standard deviation (n = 5 per group). Statistical significance was determined using one-way ANOVA followed by post-hoc Bonferroni correction. #P < 0.05 or ##P < 0.01 vs. the Control group; *P < 0.05 or **P < 0.01 vs. the TBI group; &P < 0.05 or &&P < 0.01 vs. the TBI+HG group.
Fig 5: HG increased TBI-induced apoptosis by inhibiting the MEK5/ERK5 pathway. (A) CC-3 and (D) Bax/Bcl-2 protein bands in scratched and transfected primary hippocampal cells. Bar graphs illustrate densitometric analyses of the Western blot protein bands for (B) CC-3 and (E) Bax/Bcl-2, each normalized to β-actin. Bar graphs illustrate quantitative analyses of (C) CC-3 and (F) Bax/Bcl-2 mRNA levels, each normalized to β-actin. (G) Double immunofluorescent staining of NeuN and p-MEK5. Representative confocal images stained for p-MEK5 (red) and NeuN (green) demonstrate that CA-MEK5 treatment not only increased p-MEK5 protein levels, but also markedly increased neuronal survival (scale bar, 100 μm). (H) Staining for NeuN and p-MEK5 was analyzed using MATLAB software. Data are presented as the mean ± standard deviation (n = 5 per group). #P < 0.05 vs. the Control group; *P < 0.05 or **P < 0.01 vs. the TBI group; &P < 0.05 or &&P < 0.01 vs. the TBI+HG group.
Supplier Page from Abcam for Anti-MEK5 antibody