primary antibodies against elk4  (Proteintech)

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 1. ELK4 expression is significantly reduced in IH-induced microglia and brain tissues of mice with OSA. (A) Transcriptome differences in peripheral blood mononuclear cells from 8 normal subjects and 16 patients with OSA syndrome. (B) Intersection of the human transcription factors list in the HumanTFDB database against DEGs screened in the GSE61463 dataset. (C) ELK4 mRNA expression in microglia induced with IH or not detected by RT-qPCR (t = 10.59, df = 4, p = 0.0004). (D) ELK4 protein expression in microglia induced with IH or not detected by western blot analysis (t = 31.80, df = 4, p < 0.0001). (E) ELK4 mRNA expression in the brain tissues of mice induced with OSA or not detected by RT-qPCR (t = 7.970, df = 8, p < 0.0001). (F) ELK4 protein expression in the brain tissues of mice induced with OSA or not detected by western blot analysis (t = 10.38, df = 8, p < 0.0001). For C-F, unpaired t-test. Data are expressed as the means ± SEM; n = 5; ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 2. Overexpression of ELK4 inhibits pro-inflammatory activation of microglia to ameliorate OSA-associated brain injury. (A) The mean time of escape latency during 5 days of training (F (3, 80) = 619.6, p < 0.0001), the mean percentage of time in the target quadrant (F (3, 16) = 528.4, p < 0.0001), and mean number of mouse platform crossings on day 6 (F (3, 16) = 50.13, p < 0.0001) in the MWM test. (B) The levels of IL-1β (F (3, 16) = 15.67, p < 0.0001), IL-6 (F (3, 16) = 18.99, p < 0.0001), TNF-α (F (3, 16) = 16.49, p < 0.0001), IL-10 (F (3, 16) = 15.76, p < 0.0001), TGF-β1 (F (3, 16) = 15.32, p < 0.0001), and BDNF (F (3, 16) = 18.88, p < 0.0001) in the hippocampal tissues of mice were examined using ELISA. (C) The levels of oxidative stress markers MDA (F (3, 16) = 16.74, p < 0.0001), SOD (F (3, 16) = 22.29, p < 0.0001), and GSH (F (3, 16) = 39.73, p < 0.0001) in the hippocampal tissues of mice were examined. (D) The neuronal pathomorphology in the brain tissues of mice was observed using HE staining. (E) Apoptosis in neurons in the brain tissues of mice was observed using TUNEL (F (3, 16) = 58.04, p < 0.0001). (F) Microglial activation in the brain tissues of mice was observed using immunofluorescence staining of Iba1 (F (3, 16) = 47.94, p < 0.0001). For B-F, one-way ANOVA; for A-1, two-way ANOVA. Data are expressed as the means ± SEM; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Over Expression, Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Immunofluorescence

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 3. Overexpression of ELK4 inhibits the level of inflammation and oxidative stress in BV2 cells. The BV2 cells were infected with oe-NC or oe-ELK4 and subjected to IH modeling. (A) Microglia M1/M2 phenotype was determined using flow cytometry (F (3, 8) = 80.62, p < 0.0001). (B) The levels of IL-1β (F (3, 8) = 14.37, p = 0.0014), IL-6 (F (3, 8) = 12.64, p = 0.0021), TNF-α (F (3, 8) = 16.15, p = 0.0009), IL-10 (F (3, 8) = 26.20, p = 0.0002), TGF-β1 (F (3, 8) = 23.02, P = 0.0003), and BDNF (F (3, 8) = 16.15, p = 0.0009) in BV2 cells were examined using ELISA. (C) The levels of oxidative stress markers MDA (F (3, 8) = 48.88, p < 0.0001), SOD (F (3, 8) = 45.00, p < 0.0001), and GSH (F (3, 8) = 38.03, p < 0.0001) in BV2 cells were examined. For A-C, one-way ANOVA. Data are representative of at least 3 independent experiments and presented as mean ± SEM in all assays; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Over Expression, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 4. ELK4 transcriptionally promotes FNDC5 expression by binding to the promoter of FNDC5. The correlation between ELK4 and ALDH1A3 (A), FNDC5 (B), MEPCE (C), NAA50 (D), and RSRC2 (E) in the brain tissues in the GEPIA database. (F) The binding sites between ELK4 and the FNDC5 promoter were predicted in the JASPAR website. (G) FNDC5 mRNA and protein expression in BV2 cells with different treatments were examined using RT-qPCR (F (3, 8) = 55.66, p < 0.0001) and western blot analysis (F (3, 8) = 129.1, p < 0.0001). (H) The enrichment of FNDC5 promoter fragments pulled by anti-ELK4 was examined using ChIP (t = 13.53, df = 4, p = 0.0002). (I) Luciferase activity in BV2 cells with different treatments was examined using a dual-luciferase activity assay (F (3, 8) = 72.87, p < 0.0001). (J) Irisin content in BV2 cells with different treatments was examined using ELISA (F (3, 8) = 19.79, p = 0.0005). For G and I-J, one-way ANOVA; for H, unpaired t-test. Data are representative of at least 3 independent experiments and presented as mean ± SEM in all assays; **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 5. Knockdown of FNDC5 reverses the protective effect of ELK4 on OSAS-related neuroinflammation and cognitive dysfunction. (A) The mean time of escape latency during 5 days of training (F (1, 40) = 469.4, p < 0.0001), mean percentage of time in the target quadrant (t = 21.49, df = 8, p < 0.0001), and mean number of mouse platform crossings on day 6 (t = 4.802, df = 8, p = 0.0014) in the MWM test. (B) The levels of IL-1β (t = 3.102, df = 8, p = 0.0146), IL-6 (t = 3.713, df = 8, p = 0.0059), TNF-α (t = 3.540, df = 8, p = 0.0076), IL-10 (t = 3.452, df = 8, p = 0.0087), TGF-β1 (t = 3.142, df = 8, p = 0.0138), and BDNF (t = 3.309, df = 8, p = 0.0107) in the hippocampal tissues of mice were examined using ELISA. (C) The levels of oxidative stress markers MDA (t = 3.184, df = 8, p = 0.0129), SOD (t = 4.381, df = 8, p = 0.0023), and GSH (t = 4.809, df = 8, p = 0.0013) in the hippocampal tissues of mice were examined. (D) The neuronal pathomorphology in the brain tissues of mice was observed using HE staining. (E) Apoptosis in neurons in the brain tissues of mice was observed using TUNEL (t = 4.674, df = 8, p = 0.0016). (F) Microglial activation in the brain tissues of mice was observed using immunofluorescence staining of Iba1 (t = 7.648, df = 8, p < 0.0001). (G) Irisin contents in the hippocampal tissues of mice were examined using ELISA (F (5, 24) = 27.65, p < 0.0001). For A-C and E-F, unpaired t-test; for G, one-way ANOVA; for A-1, two- way ANOVA. Data are expressed as the means ± SEM; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Activation Assay, Immunofluorescence

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 6. Schematic illustrating the mechanism. Reduced ELK4 expression in OSA reduces FNDC5 transcriptional expression, leading to pro-inflammatory activation of microglia and cognitive dysfunction. OSA progression was alleviated by activating ELK4-mediated FNDC5 transcriptional expression.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Expressing, Activation Assay