yth n 6 methyladenosine rna binding protein 2  (Proteintech)

Journal: Cancer Communications

Article Title: Helicobacter pylori CagA elevates FTO to induce gastric cancer progression via a “hit‐and‐run” paradigm

doi: 10.1002/cac2.70004

Figure Lengend Snippet: FTO increases the stability of HBEGF mRNA via YTHDF2. (A) Venn diagram showing 135 common genes among three times of repetition from MS results. (B) RIP‐qPCR verified the enrichment of HBEGF mRNA on YTHDF2 protein in MKN45 cells. (C) RNA pull‐down showing the binding of HBEGF mRNA to YTHDF2. (D) Western blotting showed an upregulated protein level of HBEGF with YTHDF2 knockdown in MKN45 cells. (E‐G) qRT‐PCR analysis of HBEGF mRNA in the indicated group when MKN45 cells were exposed to actinomycin D, with or without YTHDF2 knockdown (E), with or without FTO knockdown (F) and with or without FTO overexpression (G). (H) Expression correlation between HBEGF and YTHDF2 levels in GC tissues from the TCGA‐STAD cohort ( n = 408). (I) Log‐rank test indicating the OS of individuals with high ( n = 257) or low ( n = 618) YTHDF2 expression in the GSE62254 . (J) Western blotting showed protein level of HBEGF in MKN45 cells with FTO knockdown or YTHDF2 knockdown. (K) Schematic showing the construction of Flag‐tagged wild‐type or m 6 A modification site‐mutant HBEGF probes. (L‐N) Western blotting showed protein level of Flag‐tagged HBEGF in MKN45 cells among indicated groups, with or without FTO knockdown (L), with or without FTO overexpression (M) and with or without YTHDF2 knockdown (N). ∗ P < 0.05 and ∗∗∗ P < 0.001 according to the unpaired t ‐test (B), two‐way ANOVA (E to G), Pearson correlation tests (H) and log‐rank test (I). Abbreviations: 3’UTR, 3’untranslation region; 5’UTR, 5’untranslation region; CDS, Coding DNA Sequence; Flag‐mut, Flag‐labeled mutant HBEGF ; Flag‐WT, Flag‐labeled wild‐type HBEGF ; FTO mut , mutant FTO ; FTO , fat mass and obesity‐associated protein; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; HBEGF , heparin‐binding EGF like growth factor; MS, mass spectrometry; NC , negative control; YTHDF2, YTH N(6)‐methyladenosine RNA binding protein 2.

Article Snippet: Briefly, the cell lysates were incubated at 4°C overnight with A/G magnetic beads (Millipore) in the presence of either an IgG control antibody or a YTH N(6)‐methyladenosine RNA binding protein 2 (YTHDF2) (#24744‐1‐AP, Proteintech, Chicago, IL, USA) antibody.

Techniques: Binding Assay, Western Blot, Knockdown, Quantitative RT-PCR, Over Expression, Expressing, Modification, Mutagenesis, Sequencing, Labeling, Mass Spectrometry, Negative Control, RNA Binding Assay

Journal: Cancer Communications

Article Title: Helicobacter pylori CagA elevates FTO to induce gastric cancer progression via a “hit‐and‐run” paradigm

doi: 10.1002/cac2.70004

Figure Lengend Snippet: Graph illustration. CagA‐positive H. pylori increases FTO expression, which results in “hit‐and‐run” GC progression. However, the synergism of the FTO inhibitor MA with H. pylori eradication prevents GC development. Abbreviations: CagA, cytotoxin‐associated gene A; CEA, carcinoembryonic antigen; CK20, cytokeratin 20; CK7, cytokeratin 7; EpCAM, epithelial cell adhesion molecule; FTO, fat mass and obesity‐associated protein; FTO, fat mass and obesity‐associated protein; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; HBEGF, heparin‐binding EGF like growth factor; JUN, Jun Proto‐Oncogene; m 6 A, N6‐methyladenosine; MA, meclofenamic acid; T4SS, the type IV secretion system; YTHDF2, YTH N(6)‐methyladenosine RNA binding protein 2.

Article Snippet: Briefly, the cell lysates were incubated at 4°C overnight with A/G magnetic beads (Millipore) in the presence of either an IgG control antibody or a YTH N(6)‐methyladenosine RNA binding protein 2 (YTHDF2) (#24744‐1‐AP, Proteintech, Chicago, IL, USA) antibody.

Techniques: Expressing, Binding Assay, RNA Binding Assay