Journal: bioRxiv

Article Title: Gut sulfide metabolism modulates behavior and brain bioenergetics

doi: 10.1101/2025.04.09.647962

Figure Lengend Snippet: (A) Scheme showing experimental setup. ( B,C) Western blot analysis (B) and quantitation (C) of MT-CO1 in murine brain on 1.5% MRD. n=3 independent mice for each conditions. (D) Brain CoQ redox status in mice maintained on 1.5% MRD (n=3 independent mice). (E) Representative axial T2-weighted MRI showing asymmetric or complete loss of lateral ventricles (red arrowheads) in Villin Cre Sqor fl/fl mice compared to controls, n=4 independent mice. (F-I) Western blot (F, H) and quantitation (G, I) of the choroid plexus markers, transthyretin (TTR), and aquaporin 1 (AQP1), n=3 independent mice. (J) Scheme showing long-range modulation of brain bioenergetics and function by diet and gut SQOR oxidation capacity.

Article Snippet: MT-CO1 (1D6E1A8) and MT-CO2 (ab110258) were from Abcam and ATOH1 (21215-1-AP), transthyretin (11891-1-AP), aquaporin 1 (20333-1-AP), IBA 1 (10904-1-AP), GFAP (60190-1-Ig), CD31 (28083-1-AP), SQOR (17256-1-AP) were from Proteintech.

Techniques: Western Blot, Quantitation Assay

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Isoforms and functional domains of the chromodomain-helicase-DNA-binding protein 8 (CHD8) protein. The three isoforms of CHD8 protein, including (1) CHD8-S, a short isoform; (2) CHD8-L1, a long isoform; and (3) CHD8-L2, a long isoform . CHD8-L1 and CHD8-L2 are composed of two histone-binding chromodomains (C1 and C2, yellow), a chromatin-remodeling helicase domain (helicase, cyan), multiple protein-interacting chromatin organization modifier domains (CR, magenta), and a DNA-binding brahma and kismet domain (BRK, pink) . The position of the identified variant relative to CHD8-L1 and CHD8-L2 isoforms is indicated in red.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Functional Assay, Binding Assay, Variant Assay

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Variant validation using Sanger sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR). (a) The Sanger chromatograms indicate the absence of the variant in the maternal DNA and presence in the proband fibroblasts and blood DNA, indicating a nonmaternal inheritance of the variant. (b) Two sets of cDNA primers, including a set of primers upstream of the variant and another downstream of the variant (Table and Figure ), were used to conduct qRT-PCR on CHD8 cDNA in the proband line versus the control lines. (c) CHD8 transcripts captured by both upstream and downstream cDNA primers showed significant reduction (upstream primers: ~42%, downstream primers: ~33%) in the CHX− proband samples relative to that of controls (Wilcoxon test: p = 0.0313 for both primer sets). CHX+ samples of both the proband and the controls showed equivalent levels of CHD8 transcripts.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Variant Assay, Biomarker Discovery, Sequencing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Immunoblotting and mass spectrometry–based proteomic analysis. (a) Western blots indicating the level of CHD8 protein detected from controls (C1, C2) and proband (P) samples. Three technical repeats ( n = 3) of Western blotting using the CHD8 C-terminal antibody (Cell Signaling Technologies #11891, 1:1000) showed the relative quantities of CHD8-L1 and CHD8-L2 against GAPDH (loading control). (b) Protein band quantification of the Western blots showed a significant reduction of the CHD8-L1 and CHD8-L2 isoform levels in the proband (P) (L1: ~51%, L2: ~48%) compared to those of the controls (C) (Mann–Whitney test: p = 0.0089, p = 0.0238, respectively). (c) The abundance of CHD8 is ranked significantly lower in the proteome of the proband compared to the controls. (d) The abundance of CHD8 is significantly lower in proband fibroblasts (70%, red dot) and lies outside of the control range (80%–104%, n = 5). (e) Volcano plot showed the relative amount of proteins in the proband line compared to the controls, with vertical lines indicating +/−1.5 log 2 -fold change and the horizontal line indicating statistical significance. CHD8 is reduced significantly by ~30% ( p < 0.001) in the proband line compared to the controls. MeCP2 (green) is significantly reduced by ~43% ( p < 0.01), whereas bromodomain adjacent to zinc finger domain 1A ( BAZ1A ) encoding the accessory subunit of the ATP-dependent chromatin assembly factor (ACF) (orange) is significantly increased by ~72% ( p < 0.001). CHD8-regulated proteins (purple), including acylglycerol kinase (AGK), CDC42-binding protein kinase (CDC42BPB), phosphatase and tensin homolog (PTEN), and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), showed a reduction in their corresponding protein abundance, with AGK being the highest at ~55% ( p < 0.001). Transportin 3 (TNPO3), nuclear receptor corepressor 1 (NCOR1), and proteasome assembly chaperone 2 (PSMG2) showed an increase of abundance with TNPO3 being the highest at ~39% ( p < 0.001). (f) STRING network analysis revealed coexpression (black), interactions (magenta), and comentions in literature (lime green) between CHD8, MeCP2, CDKL5, FOXG1, and ACF.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Western Blot, Mass Spectrometry, Control, MANN-WHITNEY, Binding Assay, Phospho-proteomics, Quantitative Proteomics

Journal: Theranostics

Article Title: Growth hormone receptor disrupts glucose homeostasis via promoting and stabilizing retinol binding protein 4

doi: 10.7150/thno.61192

Figure Lengend Snippet: GHR promotes RBP4 protein homeostasis through the HIF1α/TTR axis. (A) Western blots analysis of GHR, p-SAT5 and STAT5 in the livers of AAV-infected mice as indicated. (B) Relative mRNA levels of RBP4 in the livers of AAV-infected mice as indicated (n=6). (C) The concentrations of serum RBP4 of AAV-infected mice as indicated (n=6). (D) Elution profile of chylomicrons in the serum of AAV-GFP (left) or AAV-GHR (right) mice. Purified proteins were detected in column eluents by monitoring absorbance at 280 nm. (E) Western blots of RBP4 and TTR in serum of AAV-GFP or AAV-GHR mice, which were separated by gel filtration chromatography and collected according to the ultraviolet absorption peak of fractions. (F) Western blots analysis of HIF1α in the livers of AAV-infected mice as indicated. (G) The schematic representation of the HIF1α binding site in the promoter region of TTR. (H) Western blots analysis of GHR, TTR and HIF1α in the livers of AAV-infected mice as indicated. (I) The HepG2 cells were transfected with pGL-3 or TTR promoter reporter plasmid or 5x HIF1α response elements (HRE) reporter. After transfection for 24 h, the cells were exposed to a hypoxic condition for 24 h. Then the luciferase activity was determined (n=6). (J and K) ChIP assay was performed by using anti-HIF1α antibody. The elutes were analyzed by using primers for VEGF HRE, TTR HRE, or non-HRE region. The quantitative results were obtained by real-time PCR (J, n=6) or electrophoretic assay (K). Data are expressed as the mean ± SD. ns, no significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t -test or one-way ANOVA).

Article Snippet: The primary antibodies for GHR (#sc-137185), PEPCK (#sc-32879), G6Pase (#sc-25840) (Santa Cruz Biotechnology, 1:1000), p-IR (#3024), IR (#3025), p-Akt (Ser473) (#9271), Akt(#9272), p-Foxo1 (#9464), Foxo1(#2880), p-GSK3β (#5558), GSK3β (#9315), HSL (#4107), ATGL(#2138), STAT5 (#94205), p-STAT5 (#9359) (Cell Signaling Technology, 1:1000), GAPDH (#10494-1-AP, 1:5000), PDH (#18068-1-AP), RBP4 (#11774-1-AP), TTR (#11891-1-AP), β-actin (#20536-1-AP) (ProteinTech Group, 1:1000), PDK4 (#ab38242), HIF1α (#ab228649) (Abcam, 1:1000), p-PDH (#ABS194, Merck Millipore, 1:1000) were used, and secondary antibodies including Peroxidase-AffiniPure Goat Anti-Rabbit IgG (#111-035-003) and Peroxidase-AffiniPure Goat Anti-Mouse IgG (#115-035-003) (Jackson ImmunoResearch, 1:2000-1:5000) were also used for Western blot analysis.

Techniques: Western Blot, Infection, Purification, Filtration, Chromatography, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction