Journal: Poultry Science

Article Title: Differential expressions of hypothalamic thermosensitive TRP ion channels may underlie the posthatching ontogeny of brain cooling capacity in broiler chickens

doi: 10.1016/j.psj.2023.102782

Figure Lengend Snippet: PCR primers of relevant genes used in this study.

Article Snippet: The membrane was then incubated with specific primary antibodies (1:1,000 dilution) overnight at 4°C: TRPV1 (#AF8250, rabbit polyclonal antibody from Beyotime), TRPV2 (#bs-10297R, rabbit polyclonal antibody from Beijing Biosynthesis Biotechnology Co., Ltd., China), TRPV3 (#BA2875-2, rabbit polyclonal antibody from Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), TRPV4 (#AF8253, rabbit polyclonal antibody from Beyotime), TRPA1 (#AF8241, rabbit polyclonal antibody from Beyotime), TRPM8 (#PB0882, rabbit polyclonal antibody from Boster), and GAPDH (#AF0006, mouse monoclonal antibody from Beyotime).

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Hepatitis B X-interacting protein promotes the formation of the insulin gene–transcribing protein complex Pdx-1/Neurod1 in animal pancreatic β-cells

doi: 10.1074/jbc.M117.809582

Figure Lengend Snippet: Hbxip forms a complex with Pdx-1 and Neurod1 in the insulin mini-enhancer in pancreatic β-cells. A, localization of both GFP-Hbxip and Pdx-1 was observed by confocal microscopy in the INS-1 cells. Scale bar, 25 μm for immunofluorescence. B, interaction between Hbxip and Pdx-1 was detected by co-IP assays in INS-1 cells in vivo. C, direct interaction between recombinant GST-Hbxip and His-Pdx-1 proteins was detected by GST pulldown assays followed by Western blot analysis (WB) in vitro. D and E, Pdx-1 is divided into three fragments. GST pulldown assays were performed with GST or GST fusion proteins and His tag fusion proteins containing the indicated amino acid residues of Pdx-1. The proteins were purified from bacteria by glutathione-Sepharose beads. F, INS-1 cells were transfected with Pdx-1 siRNA. ChIP assays were conducted 24 h after transfection. G, schematic showing the Ins mini-enhancer regions with Pdx-1-binding sites or negative control regions without Pdx-1-binding sites. H, interaction between Hbxip, Pdx-1, and Neurod1 was detected by co-IP assays in INS-1 cells in vivo. I, interaction of Pdx-1 with Neurod1 was detected by co-IP assays in INS-1 cells transfected with si-Control or si-Hbxip. J, interaction between Hbxip, Pdx-1, and Neurod1 was detected by co-IP assays in the primary β-cells isolated from floxed Hbxip mice. K, interaction of Pdx-1 with Neurod1 was detected by co-IP assays in the primary β-cells isolated from floxed Hbxip mice transduced with adeno-LacZ or adeno-Cre. L, interaction of Hbxip with Neurod1 was detected by co-IP assays in INS-1 cells transfected with si-Control or si-Pdx-1. M, Venn diagram shows the overlapping target genes of HBXIP and Pdx-1. N, left, real-time PCR analysis was performed to measure 10 of the 149 candidate target genes in pancreas islets of Hbxip-deficient mice and Ins2-cre (wildtype) mice; right, functional assignments for candidate target genes were verified in real-time PCR analysis. Mean ± S.D., n = 5/group. Each experiment was repeated at least three times.

Article Snippet: For Western blot analysis, the following were used: Pdx-1, primary antibody is rabbit anti-Pdx-1 (Proteintech Group, 20989-1-AP); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, A25022); Neurod1, primary antibody is mouse anti-Neurod1 (Wuhan Boster Biological Technology Ltd., BM3336); secondary antibody is IPKine HRP goat anti-mouse IgG light chain (Abbkine, A25012); Hbxip, primary antibody is rabbit anti-Hbxip (Santa Cruz Biotechnology, Santa Cruz, CA, sc-373980); and secondary antibody is IPKine HRP goat anti-rabbit IgG heavy chain (Abbkine, {"type":"entrez-nucleotide","attrs":{"text":"A25222","term_id":"904602","term_text":"A25222"}} A25222 ).

Techniques: Confocal Microscopy, Immunofluorescence, Co-Immunoprecipitation Assay, In Vivo, Recombinant, Western Blot, In Vitro, Purification, Bacteria, Transfection, Binding Assay, Negative Control, Control, Isolation, Transduction, Real-time Polymerase Chain Reaction, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Hepatitis B X-interacting protein promotes the formation of the insulin gene–transcribing protein complex Pdx-1/Neurod1 in animal pancreatic β-cells

doi: 10.1074/jbc.M117.809582

Figure Lengend Snippet: Hbxip increases the insulin expression in pancreatic β-cells through activating Pdx-1 and Neurod1. A–C, insulin levels were examined by insulin assay kit in the culture supernatant of INS-1 cells transfected with relative plasmids and siRNAs. D, interference efficiency of Neurod1 siRNA was validated by Western blot analysis in INS-1 cells. The protein levels of Neurod1 from three independent experiments were quantified using ImageJ software as shown. Mean ± S.D. Each experiment was repeated at least three times. Student's t test; *, p < 0.05; **, p < 0.01.

Article Snippet: For Western blot analysis, the following were used: Pdx-1, primary antibody is rabbit anti-Pdx-1 (Proteintech Group, 20989-1-AP); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, A25022); Neurod1, primary antibody is mouse anti-Neurod1 (Wuhan Boster Biological Technology Ltd., BM3336); secondary antibody is IPKine HRP goat anti-mouse IgG light chain (Abbkine, A25012); Hbxip, primary antibody is rabbit anti-Hbxip (Santa Cruz Biotechnology, Santa Cruz, CA, sc-373980); and secondary antibody is IPKine HRP goat anti-rabbit IgG heavy chain (Abbkine, {"type":"entrez-nucleotide","attrs":{"text":"A25222","term_id":"904602","term_text":"A25222"}} A25222 ).

Techniques: Expressing, Transfection, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Hepatitis B X-interacting protein promotes the formation of the insulin gene–transcribing protein complex Pdx-1/Neurod1 in animal pancreatic β-cells

doi: 10.1074/jbc.M117.809582

Figure Lengend Snippet: Model shows that Hbxip contributes to the modulation of insulin transcription. Hbxip acting as a co-activator promotes insulin transcription through binding to the mini-enhancer of insulin and activating transcription factor Pdx-1. Hbxip co-activates Pdx-1 through recruiting Neurod1 to Pdx-1 forming a transcription factor complex of Hbxip/Pdx-1/Neurod1. Thus, Hbxip increases the insulin expression by increasing levels of the Pdx-1/Neurod1 complex in animal pancreatic β-cells.

Article Snippet: For Western blot analysis, the following were used: Pdx-1, primary antibody is rabbit anti-Pdx-1 (Proteintech Group, 20989-1-AP); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, A25022); Neurod1, primary antibody is mouse anti-Neurod1 (Wuhan Boster Biological Technology Ltd., BM3336); secondary antibody is IPKine HRP goat anti-mouse IgG light chain (Abbkine, A25012); Hbxip, primary antibody is rabbit anti-Hbxip (Santa Cruz Biotechnology, Santa Cruz, CA, sc-373980); and secondary antibody is IPKine HRP goat anti-rabbit IgG heavy chain (Abbkine, {"type":"entrez-nucleotide","attrs":{"text":"A25222","term_id":"904602","term_text":"A25222"}} A25222 ).

Techniques: Binding Assay, Expressing

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Exosome carried NEAT1 into the cerebral cortex and NEAT1 was significantly highly expressed in sepsis-induced ferroptosis A Brain vascular permeability was detected by EBD leakage in control and model rats ( n = 10). Extracted dye contents in the formamide extracts were quantified at 620 nm; B ROS level in cerebral cortex homogenate was detected by flow cytometry. C – E ELISA analysis on Fe ion, GSH, and GPX4 levels. F HE staining on cerebral cortex in control and model rats. G Western blot analysis on serous exosome biomarkers of TSG01, CD9, and CD63. H , I The expression of NEAT1 was detected by qRT-PCR in the serous exosome (normalized to synthetic cel-miR-39-3p) and cerebral cortex (normalized to GAPDH), respectively. J The correlation analysis on the expression of NEAT1 in exosome and cerebral cortex. ** P < 0.01 vs Control; *** P < 0.001 vs Control; EBD, Evans blue dye.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Permeability, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR