Journal: Physiological Reports

Article Title: Acute tuft cell ablation in mice induces malabsorption and alterations in secretory and immune cell lineages in the small intestine

doi: 10.14814/phy2.70264

Figure Lengend Snippet: Primary antibody list.

Article Snippet: GIP , Rabbit poly , Unconjugated , Novus , NBP3‐04865 , 1:100.

Techniques: Conjugation Assay

Journal: BMB Reports

Article Title: Glucose-dependent insulinotropic polypeptide (GIP) alleviates ferroptosis in aging-induced brain damage through the Epac/Rap1 signaling pathway

doi: 10.5483/BMBRep.2024-0067

Figure Lengend Snippet: GIP overexpression facilitates cell proliferation through a well-coordinated cell cycle progression in NB41A3 cells. The (A) mRNA expression and (B) protein levels of GIP were confirmed in mock and GIP overexpressing cells using qRT-PCR and Western blotting, respectively. (C) The rate of cell proliferation was analyzed daily using CCK-8 assays until 72 h to compare proliferation between the control (Mock) and GIP overexpressing cells. (D) NB41A3 cells were collected and stained with PI to monitor cell cycle profiles for a FACS analysis. (E) A bar graph displaying the percentage of cells in each cell phase. (F–L) Real-time qPCR was used to measure the relative mRNA expression levels of cell cycle markers. Data from three independent experiments are presented as means ± SEM, and t-tests were performed to assess statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Western blotting was performed using antibodies against anti-GIP (GTX55639; GeneTex, Irvine, CA, USA), Epac (sc-28366, 1:1,000; Santa Cruz), RAP1B (NBP1-54871, 1:1,000; Novus Biologicals), xCT/SLC7A11 (MA5-35360, Thermo Fisher Scientific), Gpx-4 (sc-166570; Santa Cruz Biotechnology, Dallas, TX, USA), nuclear factor erythroid 2-related factor (Nrf2) (sc-365949), Nox1 (ab131088; Abcam), β-actin (sc-47778), and GAPDH (#5174; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Control, Staining

Journal: BMB Reports

Article Title: Glucose-dependent insulinotropic polypeptide (GIP) alleviates ferroptosis in aging-induced brain damage through the Epac/Rap1 signaling pathway

doi: 10.5483/BMBRep.2024-0067

Figure Lengend Snippet: GIP-overexpressing TG mice exhibit reduced aging-induced ferroptosis. (A) Western blotting analyses were performed to detect GIP protein expression in whole brain sections by period. (B) Proteins, including the gut, epididymis (EpiD), kidney (Kid), brain (Br), heart (He), lung (Lu), and spleen (Spl), in tissue samples from 11-week-old C57BL/6J WT mice were analyzed through western blotting. And (C) The cortex (Ctx), hippocampus (Hipp), cerebellum (CB), olfactory bulb (OB), midbrain (Mid), and medulla (Med) regions of WT mice were determined for GIP expression level. (D) A schematic showing the construction of the GIP-overexpression vector. (E) In TG mice, GIP-overexpression was confirmed using RT-PCR with primers targeting the transfected protein. (F) GIP level in mouse whole brain was analyzed in WT and GIP-Tg mice. (G) Light microscopy images of H&E-stained brain sagittal sections collected from 1-year-old mice (n = 3). (magnification: bar 100 μm). Thin arrows indicate intracerebral hemorrhages around blood vessels, and thick arrows indicate pyknotic nuclei. (H) Confocal microscopy images of cells stained with DAPI and immunostained for 4-HNE (red) in 1-year-old WT and GIP-TG mice, merged staining images are included on the right (scale bars represent 20 μm). (I) Quantification of 4-HNE positive cells (counts/mm 2 ). (J-M) mRNA levels of ferroptosis markers were assessed by qRT-PCR. (N) Protein levels of ferroptosis markers were evaluated by western blot. All data are presented as the mean ± SEM (n = 3), and t-tests were performed to evaluate statistical significance: ***P < 0.001 and ****P < 0.0001.

Article Snippet: Western blotting was performed using antibodies against anti-GIP (GTX55639; GeneTex, Irvine, CA, USA), Epac (sc-28366, 1:1,000; Santa Cruz), RAP1B (NBP1-54871, 1:1,000; Novus Biologicals), xCT/SLC7A11 (MA5-35360, Thermo Fisher Scientific), Gpx-4 (sc-166570; Santa Cruz Biotechnology, Dallas, TX, USA), nuclear factor erythroid 2-related factor (Nrf2) (sc-365949), Nox1 (ab131088; Abcam), β-actin (sc-47778), and GAPDH (#5174; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Transfection, Light Microscopy, Staining, Confocal Microscopy, Quantitative RT-PCR

Journal: BMB Reports

Article Title: Glucose-dependent insulinotropic polypeptide (GIP) alleviates ferroptosis in aging-induced brain damage through the Epac/Rap1 signaling pathway

doi: 10.5483/BMBRep.2024-0067

Figure Lengend Snippet: Activation of the Epac–Rap1b signaling pathway by GIP alleviates brain damage. Relative mRNA levels of Epac (A), Rap1a (B), and Rap1b (C) in mock and GIP-overexpressing NB41A3 cells were determined using qPCR. (D) The protein expressions of Epac and Rap1b, as detected by western blotting. (E, F) Bright-field images and its quantification in the treatment of CE3F4, glutamate, and Ferrostain-1. (G, H) Mice sagittal sections from the cerebellum of 1-year-old mice were immunofluorescence stained for GIP (red), Epac1 (green), and DAPI (blue). (I) Coronal sections showing Epac and Rap1b staining, showing notable signal increases in the TG mice (scale bars represent 100 μm). Data are presented as means ± SEM from more than three independent experiments, and t-tests were performed to assess statistical significance: **P < 0.01.

Article Snippet: Western blotting was performed using antibodies against anti-GIP (GTX55639; GeneTex, Irvine, CA, USA), Epac (sc-28366, 1:1,000; Santa Cruz), RAP1B (NBP1-54871, 1:1,000; Novus Biologicals), xCT/SLC7A11 (MA5-35360, Thermo Fisher Scientific), Gpx-4 (sc-166570; Santa Cruz Biotechnology, Dallas, TX, USA), nuclear factor erythroid 2-related factor (Nrf2) (sc-365949), Nox1 (ab131088; Abcam), β-actin (sc-47778), and GAPDH (#5174; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining