Journal: NPJ Parkinson's Disease

Article Title: Glycation modulates glutamatergic signaling and exacerbates Parkinson’s disease-like phenotypes

doi: 10.1038/s41531-022-00314-x

Figure Lengend Snippet: Wild-type littermate (WT) and Thy1-aSyn transgenic mice received an intracerebroventricular (ICV) injection of MGO or vehicle (PBS) and protein brain extracts from several regions analyzed 5 weeks post injection. Protein extracts were resolved by SDS-PAGE or loaded into membranes in a dot-blot system (see Supplementary Fig. ). Membranes were probed with anti-aSyn, anti-CEL, anti-pS129-aSyn and anti-β-actin for normalization. Representative blots showing two samples from each experimental group are shown for aSyn, and densitometric analysis represented for aSyn and CEL in a , b midbrain, c , d striatum, e , f cerebellum, g , h prefrontal cortex, and i , j hippocampus, respectively. k Representative blot showing four samples from vehicle- and MGO-injected Thy1-aSyn mice are shown for pS129-aSyn and aSyn total signal. Densitometric analysis of the ratio between pS129-aSyn and aSyn is presented. l Representative blot showing aSyn probing in soluble and insoluble fraction from vehicle- and MGO-injected Thy1-aSyn mice. Densitometric analysis of the ratio between the insoluble and the sum of both soluble and insoluble signals is presented. At least n = 5 in all groups, data in all panels are average with error bars representing standard deviation, Ordinary one-way ANOVA, * p < 0.05, ** p < 0.01, **** p < 0.0001; unpaired two-tailed t -test with equal SD, # p < 0.05.

Article Snippet: Membranes were incubated with blocking solution (5% bovine serum albumin) in 1× TBS (20 mM Tris, 136 mM NaCl, pH 7.6) at room temperature for 30 min. Primary antibody incubations were carried out overnight at 4 °C, using given concentrations in blocking solution: CEL (Mouse Anti-N ε -carboxyethyl lysine, in a dilution of 1:1000 in blocking solution, Cosmo-Bio, USA), aSyn (Purified Mouse Anti-α-Synuclein antibody, in a dilution of 1:1000 in blocking solution, BD Biosciences; San Jose, CA, USA), phosphorylated aSyn at residue 129 (pS129, [J18] SMC-600, StressMarq; Victoria, Canada), glyoxalase I (Rabbit polyclonal anti-Glyoxalase I antibody – FL-184, sc67351, Santa Cruz Biotechnology), and β-actin (Mouse Monoclonal anti-β-actin antibody in a dilution of 1:5000 in blocking solution, Ambion, Thermo Fisher Scientific; Waltham, MA, USA).

Techniques: Transgenic Assay, Injection, SDS Page, Dot Blot, Standard Deviation, Two Tailed Test

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: AT 1a influences GABAA-mediated inhibition through regulation of KCC2 expression

doi: 10.1152/ajpregu.00105.2018

Figure Lengend Snippet: Angiotensin type 1a knockdown (AT1a KD) reduced KCC2 mRNA and S940 phosphorylation in the median preoptic nucleus (MnPO). A: KCC2 mRNA was reduced in AT1a KD (n = 7) compared with scrambled sequence (Scr) (n = 7). B: there was no difference in total KCC2 protein between AT1a KD (n = 3) and Scr (n = 4). C: there was a reduction in pS940 KCC2 in AT1a KD. D: there was a reduction in the ratio of pS940 KCC2/total KCC2 compared with Scr. E: Western blots show expression of total KCC2 and pKCC2 in AT1a KD- and Scr-treated tissue. *P < 0.05.

Article Snippet: Blots were incubated in primary antibodies for Ser940 pKCC2 (1:500 rabbit polyclonal; 612-401-E15; Rockland Immunochemicals, Limerick, PA), KCC2 (1:300 rabbit polyclonal; 07-432; Millipore Sigma, Burlington, MA), GABAA-β2 (1:1,000 rabbit polyclonal; AB5561; Millipore Sigma), GABAA-β3 (1:1,000 mouse monoclonal; ab98968; Abcam, Cambridge, UK), or β-actin (1:2,000 mouse monoclonal; A5441; Sigma-Aldrich) overnight at 4°C.

Techniques: Sequencing, Western Blot, Expressing

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 1. Inhibition of micro RNA miR-122-5p on lipopolysaccharide-induced myocardial injury. Wistar rats were intravenously injected with miR-122-5p antagomir, followed by lipopolysaccharide (LPS) stimulation (n = 6 rats per group). (a-b) After LPS treatment for 12 h, heart tissues were harvested for the relative expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) using real-time quantitative PCR (RT-qPCR) or western blot assay. (c) The ratio of heart weight/ body weight (HW/BW) was calculated. (d) Hematein and eosin (H&E) staining revealed the effect of miR-122-5p on LPS-induced histopathological changes in heart. (e) Levels of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were examined by enzyme-linked immunosorbent assay (ELISA). ***p < 0.001 versus control; ##p < 0.01, ###p < 0.001 versus LPS + NC antagomir.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Inhibition, Injection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 3. In vitro analysis for beneficial role of inhibiting micro RNA miR-122-5p in lipopolysaccharide (LPS)-induced apoptosis. (a-b) Rat H9c2 cells were treated with LPS for 12 h or 24 h, and the expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were assessed by real-time quantitative PCR (RT-qPCR) or western blot analysis. (c-d) H9c2 cells were transfected with NC inhibitor or miR-122-5p inhibitor for 24 h, followed by LPS treatment for another 24 h under proper culture conditions. After that, miR-122-5p and GIT1 expression levels were measured. (e) The contents of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were detected by appropriate kits. (f) Flow cytometry showed the apoptosis of LPS-stimulated myocardial cells. (g) Western blot analysis illustrated the changes of caspase-3 expression. **p < 0.01, ***p < 0.001 versus control; ++p < 0.01, ++

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 5. Potential downstream target gene of micro RNA miR-122-5p. H9c2 cells were transfected with NC mimics, miR-122-5p mimics, NC inhibitor and miR-122-5p inhibitor for 48 h. (a-b) The expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were verified by real-time quantitative PCR (RT-qPCR) assay. (c) The predicted binding sites of miR-122-5p in the 3-UTR of GIT1, and the sequence information of miR-122-5p and GIT1 (wild- or mutant- type) was displayed. (d) Luciferase assay verified the correlation between miR-122-5p and GIT1. aap < 0.01, aaap < 0.001 versus NC mimics; bbbp < 0.001 versus NC inhibitor; ddp < 0.01 versus NC mimics + GIT1 3UTR (WT).

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 6. G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency attenuates the effects of micro RNA miR-122-5p loss on myocardial injury. (a) H9c2 cells were transfected with GIT1 siRNA to downregulate GIT1 expression. (b) The cells were transfected with GIT1 siRNA and/or miR-122-5p inhibitor, and then induced by lipopolysaccharide (LPS). GIT1 expression at mRNA and protein levels was then measured using real-time quantitative PCR (RT-qPCR) or western blot. (c) Apoptosis of myocardial cells was analyzed by flow cytometry. (d) Reactive oxygen species (ROS) production was examined using flow cytometry. (e-g) The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and tumor necrosis factor alpha (TNF-α) were assessed by the enzyme-linked immunosorbent assay (ELISA) kits. XXXp < 0.001 versus NC siRNA; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 versus LPS + miR-122-5p inhibitor + NC siRNA.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 7. G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency inhibits nuclear factor erythroid 2-related factor 2 (Nrf-2) activation. (a) Real-time quantitative PCR (RT-qPCR) assay was used to measure the heme oxygenase-1 (HO-1) and NAD(p)H: quinone oxidoreductase 1 (NQO-1) expression. (b) Nuclear Nrf-2 level was revealed using western blot analysis. ^^p < 0.01 versus LPS + miR-122-5p inhibitor + NC siRNA.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot