Journal: EMBO Reports

Article Title: CYLD-TRAF6 interaction promotes ADP-heptose-induced NF-κB signaling in H. pylori infection

doi: 10.1038/s44319-025-00480-y

Figure Lengend Snippet: ( A ) AGS cells were incubated with DMSO (vehicle) or a selective TRAF6-Ubc13 inhibitor (C25-140, 20 μM) for 6 h followed by H. pylori infection for the times shown. Total lysates were analyzed by immunoblotting using the indicated antibodies. ( B ) CYLD KO_1 AGS cells were transfected with empty vector or plasmid expressing the catalytically inactive CYLD mutant (pCYLD C601A) 48 h prior to infection with H. pylori for 30 min. Total lysates were harvested using lysis buffer containing 1% SDS (denaturing condition). IP was performed using an anti-TRAF6 antibody or isotype-matched antibody (IgG). Eluates and total lysates were analyzed by immunoblotting using the indicated antibodies. ( C ) AGS cells were incubated with DMSO (vehicle) or a selective TRAF6-Ubc13 inhibitor (C25-140, 20 μM) for 6 h followed by H. pylori infection for 30 min. Total lysates were harvested using lysis buffer containing 1% SDS (denaturing condition). IP was performed using an anti-TRAF6 antibody or isotype-matched antibody (IgG). Eluates and total lysates were analyzed by immunoblotting using the indicated antibodies. ( D ) AGS cells were left uninfected (-) or infected with H. pylori for the times shown. Total lysates were analyzed by immunoblotting using the indicated antibodies. ( E ) Following incubation of recombinant human (rh) CYLD and rhA20 proteins or rhTIFA and rhA20 in vitro, IP was performed using an antibody against A20. Eluates and input (10 ng rhCYLD, rhA20 or rhTIFA proteins) were analyzed by immunoblotting using the indicated antibodies. For ( A – D ), GAPDH serves as the loading control for the total lysates. IBs were processed in parallel and depict 1 representative of at least two independent experiments.

Article Snippet: Recombinant human A20 protein , BPS Bioscience , 80408.

Techniques: Incubation, Infection, Western Blot, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Lysis, Recombinant, In Vitro, Control

Journal: EMBO Reports

Article Title: CYLD-TRAF6 interaction promotes ADP-heptose-induced NF-κB signaling in H. pylori infection

doi: 10.1038/s44319-025-00480-y

Figure Lengend Snippet: ( A ) AGS cells were left uninfected (−) or infected with H. pylori for the times shown and harvested for total lysates. IP with an anti-TRAF6 antibody or an isotype-matched antibody (IgG) was performed. Eluates and total lysates were analyzed by immunoblotting using the indicated antibodies. ( B ) WT and A20 KO AGS cells were left uninfected or infected with H. pylori for 30 min and harvested for total lysates using lysis buffer containing 1% SDS (denaturing condition). IP with an anti-TRAF6 antibody or an isotype-matched antibody (IgG) was performed. Eluates and total lysates were analyzed by immunoblotting using the indicated antibodies. ( C ) AGS/CYLD KO_1 cells were transfected with siRNAs targeting A20 (A20 si_5 or A20 si_9 , 40 nM) or a non-targeting scrambled siRNA control (scr, 40 nM) 48 h prior to infection with H. pylori for the times shown. Total lysates were analyzed by immunoblotting using the indicated antibodies. ( D ) AGS/CYLD KO_1 cells were transfected with siRNAs targeting A20 (A20 si_5 or A20 si_9 , 40 nM) or a non-targeting scrambled siRNA control (scr, 40 nM) 48 h prior to infection with H. pylori for the times shown. Cell fractionation was performed to obtain the cytoplasmic and soluble nuclear fractions before analysis by immunoblotting using the indicated antibodies. GAPDH and C23 serve as the loading controls for the cytoplasmic and soluble nuclear fractions, respectively. For ( A – C ), GAPDH serves as the loading control for the total lysates. For all panels, IBs were processed in parallel and depict 1 representative of at least two independent experiments. .

Article Snippet: Recombinant human A20 protein , BPS Bioscience , 80408.

Techniques: Infection, Western Blot, Lysis, Transfection, Control, Cell Fractionation

Journal: EMBO Reports

Article Title: CYLD-TRAF6 interaction promotes ADP-heptose-induced NF-κB signaling in H. pylori infection

doi: 10.1038/s44319-025-00480-y

Figure Lengend Snippet: ( A ) WT and CYLD KO_1 AGS cells were left uninfected (-) or infected with H. pylori for the times shown. Total lysates were analyzed by immunoblotting using the indicated antibodies. ( B ) WT and CYLD KO_1 AGS cells were left uninfected or infected with H. pylori for the times shown. Cell fractionation was performed to obtain the cytoplasmic and soluble nuclear fractions before analysis by immunoblotting using the indicated antibodies. GAPDH and C23 serve as the loading controls for the cytoplasmic and soluble nuclear fractions, respectively. ( C ) WT and CYLD KO_1 AGS cells were left uninfected or infected with H. pylori for the times shown and harvested for total lysates. IP with an anti-TRAF2 antibody was performed. Lane 1, TRAF2 IP using lysate from H. pylori ( Hp lysate + Ab) and lane 2, total lysates from Hp 150 min treatment plus protein A/G magnetic beads (beads control) indicate the specificity of the IP and serve as controls. Eluates and total lysates were analyzed by immunoblotting using the indicated antibodies. ( D ) WT and CYLD KO_1 AGS cells were left uninfected or infected with H. pylori for 150 min. CYLD KO_1 AGS cells were transfected with recombinant human (rh) A20 proteins 24 h prior to infection by H. pylori for 150 min. Total lysates were analyzed by immunoblotting using the indicated antibodies. ( E ) A proposed model for CYLD’s role in the regulation of NF-κB signaling pathway in H. pylori infection. CYLD interacts constitutively with TRAF6, and upon H. pylori infection, this interaction stabilizes the ubiquitinylation of TRAF6. A20 negatively regulates the ubiquitinylation of TRAF6 and the ensuing classical NF-κB signaling. CYLD counters this effect, thus relieving the ‘brakes’ of A20 on classical NF-κB activation. In addition, the subsequent classical NF-κB-dependent de novo synthesis of A20 provides a negative feedback loop leading to shutdown not only of the classical but also of the alternative NF-κB pathway. For ( A , C , D ), GAPDH serves as the loading control for the total lysates. For ( A – D ), IBs were processed in parallel and depict 1 representative of at least two independent experiments. .

Article Snippet: Recombinant human A20 protein , BPS Bioscience , 80408.

Techniques: Infection, Western Blot, Cell Fractionation, Magnetic Beads, Control, Transfection, Recombinant, Activation Assay

Journal: Inorganic chemistry

Article Title: Tuning cyclometalated gold(III) for cysteine arylation and ligand-directed bioconjugation

doi: 10.1021/acs.inorgchem.1c01517

Figure Lengend Snippet: a) Schematic representation of Au(III)-mediated covalent inhibition of KRAS mutant b) Au(III) inhibitors used in this study c) KRASi-Au-2 docked with KRAS(G12C) mutant (PDB: 4LYH was used), selected structural regions with the following color scheme: Ploop (residues 10–14), teal; Cys 12, sticks colored as element color; switch-I (residues 30–40), maroon; switch-II (residues 58–72), dark pink; other regions of protein, gray; KRASi-Au-2, modeled as ball and stick.

Article Snippet: Human recombinant K-Ras(G12C) (N-terminal His - tag) protein was purchased from BPS Bioscience, USA.

Techniques: Inhibition, Mutagenesis

Journal: Inorganic chemistry

Article Title: Tuning cyclometalated gold(III) for cysteine arylation and ligand-directed bioconjugation

doi: 10.1021/acs.inorgchem.1c01517

Figure Lengend Snippet: KRAS(G12C) recombinant protein was labelled with KRASi-Au-3 A) Extracted Ion Chromatogram (XIC) for the KRAS(G12C) labelled with KRASi-Au-3 B) Base peak of the MS/MS spectrum confirmed the modification of G12C position (m/z = 524.2464, z=3 and 785.8649. z=2) C) LC-MS/MS of the KRASi-Au-3 modified KRAS(G12C) peptide (residue 6-16)

Article Snippet: Human recombinant K-Ras(G12C) (N-terminal His - tag) protein was purchased from BPS Bioscience, USA.

Techniques: Recombinant, Tandem Mass Spectroscopy, Modification, Liquid Chromatography with Mass Spectroscopy

Journal: Inorganic chemistry

Article Title: Tuning cyclometalated gold(III) for cysteine arylation and ligand-directed bioconjugation

doi: 10.1021/acs.inorgchem.1c01517

Figure Lengend Snippet: a) Viability (MTT assay) of KRAS(G12C)-mutant cell lines MiaPaCa (pancreas, homozygous G12C), H358 (lung, heterozygous G12C), H460 (lung, Q61H mutation) after treatment with 1, KRASi-Au-1, KRASi-Au-2, and KRASi-Au-3 for 72 h at 0 −100 μM ; b) Representative bar graph illustrating IC50 values of complexes 1, KRASi-Au-1, KRASi-Au-2, and KRASi-Au-3. (n=3 biological replicates, error bars denoted mean ± s.e.m) c) Cellular uptake of KRASi-Au-1, KRASi-Au-2 and KRASi-Au-3 in MiaPaCa cell, ****. p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: Human recombinant K-Ras(G12C) (N-terminal His - tag) protein was purchased from BPS Bioscience, USA.

Techniques: MTT Assay, Mutagenesis