Journal: Communications Biology

Article Title: Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy

doi: 10.1038/s42003-022-04021-4

Figure Lengend Snippet: a Myofilament from Ntg ( n = 3) and ErbB2 ( n = 3) transgenic mice hearts were freshly isolated on ice-cold buffers containing Proteinase and Phosphatase Inhibitors Cocktails (Roche). b All material was resuspended in TEAB, then reduced and alkylated. Tryptic peptides were desalted and labeled with 6-plex isobaric tandem mass tags (TMT). c The digested and labeled peptides were pooled and desalted with C 18 SEP-PAK. The enrichment for phosphotyrosine was performed with PTMScan Phospho-Tyrosine Rabbit mAb ( P -Tyr-1000) kit (Cell Signaling Technology). The eluted peptide samples were desalted using C18 STAGE tips d Easy-nanoLC 1200 nanoflow liquid chromatography system coupled to Orbitrap Fusion Lumos Tribrid.

Article Snippet: The digested and labeled peptides were pooled and desalted with C 18 SEP-PAK (Waters), followed by pTyrsoine enrichment using PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) kit (Cell Signaling Technology), see Fig. .

Techniques: Transgenic Assay, Isolation, Labeling, Liquid Chromatography

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: Verification of DDIT4 expression and H pylori colonization in the gastric tissues of Ddit4 +/+ and Ddit4 -/- mice. ( A ) The qRT-PCR analysis indicating the mRNA levels of DDIT4 in the stomach tissues of Ddit4 +/+ and Ddit4 -/- mice. ( B ) The ratio of p-mTOR to total mTOR protein levels in infected WT and Ddit4-knockout mice. ( C ) Silver staining showing H pylori colonization in the gastric tissues of mice (Scale bar, 10 μm). ( D ) Colony-forming units (CFUs) per gram stomach in animals were evaluated for H pylori colonization by culture at 4 days after sacrifice. ( E ) IHC scores of ATP4A and ATP4B in infected gastric tissues of Ddit4 +/+ and Ddit4 -/- mice. ( F ) Immunofluorescence staining for MUC2 ( left panel ) or FABP1 ( right panel ) on infected gastric tissues from Ddit4 +/+ and Ddit4 -/- mice (Scale bar, 20 μm). ns, no significance. ∗ P < .05; ∗∗ P < .01; and ∗∗∗ P < .001 were considered significant.

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Expressing, Quantitative RT-PCR, Infection, Knock-Out, Silver Staining, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: Ddit4 deletion results in gastric metaplasia in animal model of H pylori pathogenesis. ( A ) Western blot analysis verifying the loss of DDIT4 and activation of p-mTOR in gastric tissues of Ddit4 -/- mice. ( B ) Histopathology of gastric tissues of Ddit4 +/+ (n = 8) and Ddit4 -/- (n = 8) mice with or without H pylori infection (Scale bar, 10 μm). ( C ) Gastric pathology scores from the mice in panel B. ( D ) PAS staining showing prominent mucous cell metaplasia in gastric tissues (Scale bar, 10 μm). ( E ) Immunofluorescence staining for the expression of Ki67 in gastric tissues of WT ( Ddit4 +/+ ) and Ddit4 -/- mice with or without H pylori infection (Scale bar, 10 μm). ( F ) IHC staining for ATP4A and ATP4B expression in the gastric tissues of H pylori -infected Ddit4 +/+ and Ddit4 -/- mice (Scale bar, 10 μm). ( G ) The Western blot analysis showing the protein levels of ATP4A and ATP4B in gastric tissues of infected Ddit4 +/+ and Ddit4 -/- mice. ( H ) Immunofluorescence staining for GIF and GSII on the gastric tissues from H pylori -infected Ddit4 +/+ and Ddit4 -/- mice (Scale bar, 50 μm). ( I ) Immunofluorescence staining for CD44V9 on gastric tissues of Ddit4 +/+ and Ddit4 -/- mice infected with H pylori. (Scale bar, 20 μm). ∗P < .05; ∗∗ P < .01; and ∗∗∗ P < .001 were considered significant.

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Animal Model, Western Blot, Activation Assay, Histopathology, Infection, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: Clinical relevance of DDIT4 and ferroptosis in human gastric intestinal metaplasia tissues. ( A ) Western blot data for the expression levels of DDIT4, ALOX15, HMOX1 and GPX4 in human GIM and non-atrophic gastritis (GS) tissues. ( B–C ) IHC staining showing DDIT4 and GPX4 expression in human GS and GIM tissues. Representative images (Scale bar, 10 μm) shown in ( B ). Quantification of staining shown in ( C ). ( D–E ) C11-BODIPY and DCFH-DA staining measured with a fluorescence microscope showing lipid peroxidation ( D ) and ROS levels ( E ) in human GS and GIM tissues. ( F ) Diagram of the mechanism by which loss of DDIT4 promoted H pylori -induced gastric metaplasia lesions through metabolic regulation of ferroptosis. Chronic H pylori infection causes the loss of DDIT4 and mTOR signaling pathway activation, which in turn inhibits GSH synthesis and increases ROS levels and lipid peroxides, inducing ferroptosis in the gastric microenvironment. As a result, gastric metaplasia lesions develop following parietal cell loss. ∗ P < .05; ∗∗ P < .01; and ∗∗∗ P < .001 were considered significant.

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Fluorescence, Microscopy, Infection, Activation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Protective Role of DDIT4 in Helicobacter pylori -induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

doi: 10.1016/j.jcmgh.2024.101448

Figure Lengend Snippet: The Primary Antibodies Used in This Study

Article Snippet: p-mTOR , Cell Signaling Technology , #5566 , WB , 1: 1000.

Techniques: Immunohistochemistry-IF