Journal: Frontiers in Physiology

Article Title: Chronic fetal hypoxia and antenatal Vitamin C exposure differentially regulate molecular signalling in the lung of female lambs in early adulthood

doi: 10.3389/fphys.2024.1488152

Figure Lengend Snippet: Effect of chronic fetal hypoxia and antenatal Vitamin C on the structure of the lung in young adult lamb. Micrographs (Scale bar = 50 μm) demonstrating no primary antibody negative control (A) , 1:500 rabbit serum negative control (B) , SP-B immunoreactivity (brown intracellular precipitate) in the alveolar epithelium in the lung of Normoxic + Saline [ (C) ; N, blue bars], Normoxic + Vitamin C [ (D) ; NVC, blue hashed bars], Hypoxic + Saline [ (E) ; H, red bars], Hypoxic + Vitamin C [ (F) ; HVC, red hashed bars] lambs. Numerical density of SP-B cells is expressed as mean ± SEM (G) . Expression of ELN gene [ (H) ; presented as mRNA mean normalized expression (MNE) ± SEM] and elastin protein [ (I) ; presented as mean protein expression in arbitrary units (AU) ± SEM; 68 kDa band] in the postnatal lung. Western blot image represents target protein (J) and reference protein (Vinculin, 120 kDa band) obtained from the same gel. ⊗ = Outlier ± 2 SD from the treatment group mean not included in analysis for this study or excluded due to technical error in the Western blot procedure (last well). Data were analyzed by Two-Way ANOVA for main effects (treatment or drug) and interaction (treatment x drug). P < 0.05 was considered statistically significant. # = effect of treatment (Normoxic vs. Hypoxic) and * = effect of drug (Saline vs. Vitamin C). All significant results presented are main effects.

Article Snippet: Total target protein abundance was then normalized to Ponceau S (total protein stain) or reference protein Vinculin (1:2000, in 5% BSA IN TBST, XP HRP conjugate, #18799S, Cell Signalling Technology; 140 kDa band).

Techniques: Negative Control, Saline, Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Chronic fetal hypoxia and antenatal Vitamin C exposure differentially regulate molecular signalling in the lung of female lambs in early adulthood

doi: 10.3389/fphys.2024.1488152

Figure Lengend Snippet: Effect of chronic fetal hypoxia and antenatal Vitamin C on expression of vasodilator pathway and oxidative stress markers [ HMOX-1 (A) and NOX-4 (B) ], antioxidant defence [ CAT gene (C) and Catalase protein [ (D) ; 60 kDA band on blot in (H) ]], regulators of hypoxia signalling and feedback [ HIF-2α (E) , EGLN-2 gene (F) and PHD-1 protein (G) ; 44 kDa band; band 2 on blot in (I) ] in the lung of young adult lambs. Data expressed as mRNA mean normalized expression (MNE) or mean protein expression in arbitrary units (AU) ± SEM. Normoxic (N) in blue bars and Hypoxic (H) in red bars. Saline exposed lambs in open bars (N and H) and Vitamin C exposed lambs (NVC and HVC) in hashed bars. Western blot images (H, I) represent target protein and reference protein for Saline (S) and Vitamin C (VC) lambs in Normoxic (N) and Hypoxic (H) groups. ⊗ = Outlier ± 2 SD from the treatment group mean not included in analysis for this study or excluded due to technical error in the Western blot procedure (last well). Reference protein Vinculin ( H, I ; 120 kDa band) is obtained from the same gel. Data were analyzed by Two-Way ANOVA for main effects (treatment or drug) and interaction (treatment x drug). When a significant interaction was detected, data was split to determine the effect of drug administration in Normoxic and Hypoxic lambs independently using the Students’ unpaired t -test. P < 0.05 was considered statistically significant. # = effect of treatment (Normoxic vs. Hypoxic) and * = effect of drug (Saline vs. Vitamin C). All significant results presented are main effects, with the exception of CAT and HIF-2α both of which had a significant interaction and further analysis determined significant effect of Vitamin C in the lung of H lambs only.

Article Snippet: Total target protein abundance was then normalized to Ponceau S (total protein stain) or reference protein Vinculin (1:2000, in 5% BSA IN TBST, XP HRP conjugate, #18799S, Cell Signalling Technology; 140 kDa band).

Techniques: Expressing, Saline, Western Blot

Journal: Frontiers in Physiology

Article Title: Chronic fetal hypoxia and antenatal Vitamin C exposure differentially regulate molecular signalling in the lung of female lambs in early adulthood

doi: 10.3389/fphys.2024.1488152

Figure Lengend Snippet: Effect of chronic fetal hypoxia and antenatal Vitamin C on expression of factors regulating glucocorticoid availability [ HSD11B1 gene, (A) ; 11βHSD-1 protein, (B) , 40 kDa band on blot (F) ], surfactant lipid transport [ ABCA3 gene (C) ; ABCA3 protein (D) , 130 kDa band on blot (G) ] and inflammation [ IL-1B gene, (E) ] in the lung of young adult lambs. Data expressed as mRNA mean normalized expression (MNE) or mean protein expression in arbitrary units (AU) ± SEM. Normoxic (N) in blue bars and Hypoxic (H) in red bars. Saline exposed lambs in open bars (N and H) and Vitamin C exposed lambs (NVC and HVC) in hashed bars. Western blot images represent target protein and reference protein for Saline (S) and Vitamin C (VC) lambs in Normoxic (N) and Hypoxic (H) groups. ⊗ = Outlier ± 2 SD from the treatment group mean not included in analysis for this study or excluded due to technical error in the Western blot procedure (last well). Reference protein Vinculin (120 kDa band) is obtained from the same gel that was used to determine expression of both 11βHSD-1 (F) and ABCA3 (G) . Data were analyzed by Two-Way ANOVA for main effects (treatment or drug) and interaction (treatment x drug). When a significant interaction was detected, data was split to determine the effect of drug administration in Normoxic and Hypoxic lambs independently using the Students’ unpaired t -test. P < 0.05 was considered statistically significant. # = effect of treatment (Normoxic vs. Hypoxic). * = effect of drug (Saline vs. Vitamin C). All significant results presented are main effects.

Article Snippet: Total target protein abundance was then normalized to Ponceau S (total protein stain) or reference protein Vinculin (1:2000, in 5% BSA IN TBST, XP HRP conjugate, #18799S, Cell Signalling Technology; 140 kDa band).

Techniques: Expressing, Saline, Western Blot

Journal: Scientific Reports

Article Title: Nesfatin-1-Like Peptide Encoded in Nucleobindin-1 in Goldfish is a Novel Anorexigen Modulated by Sex Steroids, Macronutrients and Daily Rhythm

doi: 10.1038/srep28377

Figure Lengend Snippet: ( a ) Schematic representation of the NUCB1 precursor showing the signal peptide, and nesfatin-1-like peptide (1–77 amino acids) regions. Nesfatin-1-Like Peptide. 2/3 is referred as NLP 2/3. The alignment of NLP sequences from various species is shown underneath. Phylogenetic analysis of nucleobindin-1 gene sequences of various species is shown in (b) . NUCB1 sequences consisting of signal peptide (1–24 amino acids) and the putative bioactive core (24–53 amino acids) were used for generating the cladogram. GenBank accession numbers of sequences used are: Carassius auratus ( KU903286 ), Neolamprologus brichardi* (XM_006803054.1), Drosophila melanogaster (NM_140751.4), Poecilia formosa* (XM_007562129.1), Xenopus tropicalis (NM_213689.2) Danio rerio (NM_001045463.1), Mus musculus (NM_001163662.1), Rattus norvegicus (NM_053463.1), Homo sapiens (NM_006184.5). Asterisk (*) associated with species names denotes predicted NUCB1 sequences. (c) The data was quantified using RT-qPCR (Real-Time - Quantitative PCR) in goldfish. The mRNA expression was normalized to elongation factor (EF)-1α (n = 6 goldfish). Western blot showing NUCB1 in brain, pituitary, gut, ovary and testis (d) , preabsorption control showing no immunoreactivity in tissues tested (e) , and vinculin (f) (n = 4 goldfish, representative blot is shown). Asterisk denotes significant differences (p < 0.05). Data are represented as mean + SEM. One-way ANOVA (non-parametric) followed by Tukey’s multiple comparison test were used for statistical analysis.

Article Snippet: In order to detect the presence of NUCB1 and vinculin (reference) protein, rabbit polyclonal anti-mouse nucleobindin-1 (custom synthesized, Catalog # 1312- PAC- 02, 1:3000, Pacific immunology, Ramona, CA) for NUCB1 and rabbit polyclonal anti-vinculin (Catalog # ab73412, 1:1000, Abcam, Massachusetts) for vinculin were used.

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Comparison