Journal: Viruses

Article Title: Recombinant Pseudorabies Virus with TK/gE Gene Deletion and Flt3L Co-Expression Enhances the Innate and Adaptive Immune Response via Activating Dendritic Cells

doi: 10.3390/v13040691

Figure Lengend Snippet: Characterization of the recombinant HNX-TK − /gE − -Flt3L. ( A ) Detection of the transcription of the Flt3L gene in HNX-TK − /gE − -Flt3L virus- and HNX-infected cells through RT-qPCR (**** < 0.001). ( B ) Detection of the Flt3L protein and viral gB protein expression through an immunofluorescence assay (IFA). Green: gB-positive cells; red: Flt3L-positive cells; blue: DAPI-stained PK-15 cell nucleus. DAPI, 4’,6-diamidino-2-phenylindole. ( C ) Western blot analysis of β-actin, gD, and Flt3L of the PK-15 cells infected with HNX-TK − /gE − -Flt3L, HNX, or DMEM as a control. ( D ) One step growth curves of HNX-TK − /gE − -Flt3L and HNX in PK-15 cells.

Article Snippet: The blots were blocked by skim milk (BD, Bioscience, Inc., Saint Louis, MO, USA), and this was followed by incubation with rabbit anti-Flt3L polyclonal antibody (Bioss, Beijing, China), mouse anti-gD monoclonal antibody (Keqian Ltd., Wuhan, China), and mouse anti-β-actin monoclonal antibody (Bioss).

Techniques: Recombinant, Infection, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Neuroendocrinology

Article Title: Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

doi: 10.1159/000521236

Figure Lengend Snippet: Effects of DHT on insulin and leptin signaling in the hypothalamus of 6-week-old, prepubertal, nonobese rats. a, e Representative immunoblots showing phosphorylated and total IR, AKT, GSK, JAK2, and Stat3 levels, with β-actin as the loading control. b–d, f, g Densitometry analysis and comparison of phosphorylated forms versus total amounts of different proteins. DHT treatment significantly decreased the phosphorylation levels of these signaling proteins. h Expression levels of feeding-related genes in the hypothalamus of 6-week-old DHT and control rats. Note the significant increase in the mRNA levels of NPY and Agrp genes. Experiments were performed in 3 rats for each group. Data are expressed in mean ± SEM ( n = 6). * p < 0.05, ** p < 0.01. SEM, standard error.

Article Snippet: β-Actin , Abcam , ab8226 , 1:10,000.

Techniques: Western Blot, Expressing

Journal: Neuroendocrinology

Article Title: Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

doi: 10.1159/000521236

Figure Lengend Snippet: Effect of DHT on insulin and leptin signaling in the hypothalamus of 7-week-old pair-fed rats. a, f Representative immunoblots showing phosphorylated and total IR, AKT, GSK, JAK2, and Stat3 levels, with β-actin as the loading control. b–e, g–j Densitometry analysis and comparison of phosphorylated versus total amounts of proteins. DHT treatment significantly decreased the phosphorylation levels of these signaling factors after pair feeding. Experiments were performed in 3 rats for each group. Data are expressed in mean ± SEM. * p < 0.05, ** p < 0.01. SEM, standard error.

Article Snippet: β-Actin , Abcam , ab8226 , 1:10,000.

Techniques: Western Blot

Journal: Neuroendocrinology

Article Title: Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

doi: 10.1159/000521236

Figure Lengend Snippet: Effect of central leptin on the hypothalamus. a Leptin levels in the CSF were significantly decreased in DHT rats than in control rats. b ICV injection of leptin inhibited the improvement of food intake caused by DHT. c Representative immunoblots showing phosphorylated and total Stat3 levels, with β-actin as the loading control. d Densitometry analysis and comparison of phosphorylated forms versus total amounts of proteins. The phosphorylation of STAT3 was also increased in the DHT rats. Data were expressed as mean ± SEM. * p < 0.05. SEM, standard error.

Article Snippet: β-Actin , Abcam , ab8226 , 1:10,000.

Techniques: Injection, Western Blot

Journal: The Journal of cell biology

Article Title: Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

doi: 10.1083/jcb.202305048

Figure Lengend Snippet: Figure 1. Transcriptional reprogramming of neuronal metabolism during glucose deprivation. (A) Schematic for glucose deprivation of rat cortical neurons. A subset of ∼250 genes significantly upregulated during glucose deprivation (P value <0.01) were selected for pathway analysis using annotated gene sets from the Molecular Signatures Database (MSigDB). n = 3 cortical samples from one rat litter. (B) Enrichment of genes in MSigDB Reactome pathways with adjusted P value <0.05. (C) Enrichment of the target genes of the CREB transcription factor in glucose-deprived neurons (adjusted P value <0.05). The target gene Pgc1α (PPARGC1A) is highlighted in red. (D) Immunostaining of cortical neuronal cultures (treated as in A) with an anti-phospho-CREB antibody and Hoechst nuclear stain. Arrowheads denote pCREB-positive nuclei. (E) Fraction of nuclei with positive p-CREB staining in fields of view (FOV), determined as described in Materials and methods. % total ± SEM: +glucose (3 h), 14.87 ± 2.29, −glucose (3 h), 27.41 ± 3.3. n = 17–22 FOVs. (F) Relative mRNA expression of Pgc1α in neuronal cultures treated as in A, with or without the AMPK inhibitor, dorsomorphin. Values are normalized to β-actin mRNA and expressed relative to the +glucose condition. Average normalized mRNA level ± SEM: −glucose (1 h), 2.83 ± 0.42; −glucose (3 h), 3.67 ± 0.46; −glucose + dorsomorphin (3 h), 1.32 ± 0.22. n = 3–10 cortical samples. Bar graphs are plotted as mean ± SEM. Mann–Whitney U test (E), one-way ANOVA (F). See Table 1.

Article Snippet: The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/ HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat).

Techniques: Immunostaining, Staining, Expressing, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

doi: 10.1083/jcb.202305048

Figure Lengend Snippet: Figure 2. Glucose deprivation stimulates neuronal Sirt3 expression and deacetylation of mitochondrial proteins. (A) Relative Sirt3 mRNA expression in control and glucose-deprived neu- rons. Values are normalized to β-actin mRNA and expressed relative to control (+glucose). n = 3–10 cortical samples. Average normalized mRNA level ± SEM: −glucose (1 h), 2.58 ± 0.62; −glucose (3 h), 2.20 ± 0.27; −glucose + dorsomorphin (3 h), 1.12 ± 0.08. n = 3–11 cortical samples. (B) Relative Sirt3 mRNA expression in cultures transduced with adenoviral particles encoding GFP (control) and GFP-PGC1α, normalized to 18s rRNA and ex- pressed relative to control. Average normalized mRNA level ± SEM: GFP-PGC1α, 1.93 ± 0.30. n = 16 cortical samples/condition. (C) Immunoblotting of Sirt3 protein expression in cortical neurons with antibodies against Sirt3 and the cytosolic and mitochondrial controls, β-actin, and ATPase5β, respectively. (D) Sirt3 band intensity normalized to the ATPase5β and expressed relative to control. Average normalized Sirt3 band intensity ± SEM: +glucose, 0.99 ± 0.05; −glucose, 1.74 ± 0.16. n = 7 cortical samples/condition. (E) A paradigm for the analysis of Sirt3 expression in hippocampi of mice fed ad libitum (ad lib) or alternate-day fasted for 6 mo (ADF). Schematic created with https:// biorender.com. (F) Immunoblotting of Sirt3 protein in mouse hippocampal lysates. (G) Sirt3 band intensity normalized to the ATPase5β band and expressed relative to the ad lib mice. Average normalized Sirt3 band intensity ± SEM: ad lib, 1 ± 0.24; ADF (6 mo), 1.88 ± 0.19. n = 6 mice/condition. (H) Mitochondrial and cytosolic fractions isolated from cortical neuronal cul- tures maintained for 3 h with (+) or without (−) glucose. High and low exposures were 60 and 25 s, respectively. (I) Intensity of lysine acetylation bands normalized to α-tubulin or ATPase5β plot- ted relative to control. Average normalized Ac-K intensity: cytosolic fraction (−glucose), 1.21 ± 0.15; mitochondrial fraction (−glucose), 0.66 ± 0.08. n = 5 cortical samples. One-way ANOVA (A), two- tailed, unpaired t test (B), Mann–Whitney U test (D and G), one sample t test (I). See Table 1. Source data are available for this figure: SourceData F2.

Article Snippet: The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/ HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat).

Techniques: Expressing, Control, Transduction, Western Blot, Isolation, Two Tailed Test, MANN-WHITNEY