Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) Schematic diagram showing the experimental procedure of the ICH rat. (b) Forelimb placing test and corner turn test of rats. (c) Brain water content of rats. (d) Real-time PCR and western blot were used to verify the expression of UBL4A in brain tissue. (e) Typical hematoxylin-eosin (HE) staining images presenting the striatal hematomas in rats. Scale bar: 500 µ m (left) and 100 µ m (right). (f) Fluoro-Jade B (FJB) staining was performed to detect neuronal cell death in rats. Scale bar: 50 µ m. (g) Co-localization of ubiquitin-like protein 4A (UBL4A) and NeuN was detected by immunofluorescence staining. Scale bar: 50 µ m. ICH, intracerebral hemorrhage. * P <0.05, ** P <0.01, ns: no significance.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Ubiquitin Proteomics, Immunofluorescence

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) The diagram depicts the flow chart of the cell experiment. (b) Real-time PCR and western blot were used to verify the expression level of ubiquitin-like protein 4A (UBL4A) in neurons. (c) CCK-8 assay was performed to determine cell viability. (d) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µ m. LV-shNC, negative control shRNA; LV-shUBL4A, shUBL4A knockdown; LV-vector, empty vector; LV-UBL4A, UBL4A overexpression. * P <0.05, ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Ubiquitin Proteomics, CCK-8 Assay, TUNEL Assay, Staining, Negative Control, shRNA, Knockdown, Plasmid Preparation, Over Expression

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10 3 ). (b) Manganese superoxide dismutase (MnSOD) activity was measured with MnSOD assay kit. (c) Detection of mitochondrial membrane potential by JC-1 staining. (d) The relative mitochondrial DNA (mtDNA) level was detected by real-time PCR. LV-shNC, negative control shRNA; LV-shUBL4A, shUBL4A knockdown; LV-vector, empty vector; LV-UBL4A, UBL4A overexpression. * P <0.05, ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Flow Cytometry, Activity Assay, Membrane, Staining, Real-time Polymerase Chain Reaction, Negative Control, shRNA, Knockdown, Plasmid Preparation, Over Expression

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) Real-time PCR to verify the expression of miR-34a-5p in neurons. (b) Real-time PCR and western blot were used to verify the expression level of ubiquitin-like protein 4A (UBL4A) in neurons with miR-34a-5p knockdown. (c) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µ m. (d) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10 3 ). LV-shNC, negative control shRNA; LV-miR-34a-sponge, miR-34a-5p knockdown. ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Ubiquitin Proteomics, Knockdown, TUNEL Assay, Staining, Flow Cytometry, Negative Control, shRNA

Journal: Experimental Animals

Article Title: Ubiquitin-like 4A alleviates the progression of intracerebral hemorrhage by regulating oxidative stress and mitochondrial damage

doi: 10.1538/expanim.24-0035

Figure Lengend Snippet: (a) Predicted miR-34a-5p target sequences in 3′-UTR of Ubl4a (Left). Dual-luciferase reporter assay was conducted to verify whether ubiquitin-like protein 4A (UBL4A) was the target of miR-34a-5p (Right). NC mimics, negative control mimics; WT, wild type; MUT, mutant type. (b) Western blot was used to verify the expression level of UBL4A in neurons. (c) TUNEL staining was used to detect cell apoptosis. Scale bar: 50 µ m. (d) The level of reactive oxygen species (ROS) in cells was determined by flow cytometry using a dichlorodihydrofluorescein diacetate (DCFH-DA) based kit (cell number: ×10 3 ). LV-shNC, negative control shRNA; LV-miR-34a-sponge, miR-34a-5p knockdown. LV-shUBL4A, shUBL4A knockdown. * P <0.05, ** P <0.01.

Article Snippet: Tissue sections were blocked with UBL4A (1:100; 14253-1-AP; Proteintech, Wuhan, China) and Neuronal Nucleus antibody (NeuN; 1:300; ab104224; Abcam, Shanghai, China) overnight at 4°C, followed by FITC-labeled goat anti-rabbit IgG (1:200; ab6717; Abcam) and Cy3-labeled goat anti-mouse IgG (1:200; A-21424; Invitrogen, Carlsbad, CA, USA) were applied in a dark box for 90 min. After rinsing with PBS, 4’,6-diamidino-2-phenylindole (DAPI; Aladdin, Shanghai, China) was used as a counterstain.

Techniques: Luciferase, Reporter Assay, Ubiquitin Proteomics, Negative Control, Mutagenesis, Western Blot, Expressing, TUNEL Assay, Staining, Flow Cytometry, shRNA, Knockdown