Journal: Investigational new drugs

Article Title: The up-regulated expression level of deubiquitinating enzyme USP46 induces the apoptosis of A549 cells by TRAF6.

doi: 10.1007/s10637-025-01532-9

Figure Lengend Snippet: Fig. 2 The interaction between USP46 and TRAF6. A and B Western blot analysis of TRAF6 expression in normal pneu- monocyte and lung cancer cell lines. C and D A549 cells were transfected with His-USP46 and Flag-TRAF6 in the indicated combinations. Immunoprecipi- tation was carried out to explore the interaction between USP46 and TRAF6

Article Snippet: Antibodies against TRAF6 (cat. no. #8028 s), AKT (cat. no. #4691), phosphorylation-AKT (cat. no. #4060), mTOR (cat. no. #2983), phosphorylation-mTOR (cat. no.#5536), ubiquitin (1:1000), Caspase- 9 (cat. no.7237), Caspase- 3 (cat. no.14220,), Bax (cat. no. #41,162), Bcl- 2 (cat. no. #15,071), GADPH (cat. no.5174) were purchased from Cell Signaling Technology (CST, USA).

Techniques: Western Blot, Expressing, Transfection

Journal: Investigational new drugs

Article Title: The up-regulated expression level of deubiquitinating enzyme USP46 induces the apoptosis of A549 cells by TRAF6.

doi: 10.1007/s10637-025-01532-9

Figure Lengend Snippet: Fig. 3 USP46 inhibited the ubiquitination of TRAF6 and stabilized the expression of TRAF6. A Overexpression of USP46 affected the ubiquitina- tion of TRAF6. Cells in each group were treated with the proteasomal inhibitor MG132. Cell lysates were collected and subjected to immuno- precipitation with the anti- TRAF6 antibody. The levels of ubiquitin-conjugated TRAF6 were detected by Western blot. B Representative Western blot and relative expression of TRAF6 in a graph revealed that USP46 overexpression inhibits degeneration of TRAF6 protein in A549 cells

Article Snippet: Antibodies against TRAF6 (cat. no. #8028 s), AKT (cat. no. #4691), phosphorylation-AKT (cat. no. #4060), mTOR (cat. no. #2983), phosphorylation-mTOR (cat. no.#5536), ubiquitin (1:1000), Caspase- 9 (cat. no.7237), Caspase- 3 (cat. no.14220,), Bax (cat. no. #41,162), Bcl- 2 (cat. no. #15,071), GADPH (cat. no.5174) were purchased from Cell Signaling Technology (CST, USA).

Techniques: Ubiquitin Proteomics, Expressing, Over Expression, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Comprehensive Atomic-Scale 3D Viral-Host Protein Interactomes Enable Dissection of Key Mechanisms and Evolutionary Processes Underlying Viral Pathogenesis

doi: 10.1101/2025.03.28.645946

Figure Lengend Snippet: (A) Schematic of RIG-I-like receptor and Herpes simplex virus-1 UL37 signaling. (B) Model demonstrating shared interface residues on TRAF6 for UL37 (predicted by Alphafold) and MAVS (PDB: 4Z8M) binding. (C) Schematic of experiments investigating UL37 suppression of MAVS signaling. 293T MAVSKO IFNB1-GLuc reporter cells induce Gaussia luciferase (GLuc) driven by tandem IFNB1 promoter elements after MAVS overexpression. (D) (Top) Relative GLuc in the supernatant (S/N) from 293T MAVSKO IFNB1-GLuc cells following overexpression of Flag-MAVS and increasing doses of UL37-V5 at 36 hours post-transfection (hpt). (Bottom) Immunoblot analysis of lysates from cells treated in the indicated manner. (E) (Top) Relative GLuc in the S/N from 293T MAVSKO IFNB1-GLuc cells following overexpression of Flag-MAVS and UL37-V5 WT or C819S deamidation mutant at 36 hpt. (Bottom) Immunoblot analysis of lysates from cells treated in the indicated manner. (F) Immunoblot analysis of anti-Flag and anti-HA immunoprecipitated extracts as well as input lysates from 293T MAVSKO cells transfected with HA-TRAF6, Flag-MAVS, and UL37-V5 as indicated (24 hpt). Quantification of HA-TRAF6 relative to immunoprecipitated Flag-MAVS and Flag-MAVS relative to immunoprecipitated HA-TRAF6 are shown below as indicated. (G) (Top) Immunoblot analysis of anti-V5 immunoprecipitated extracts and input lysates from 293T PRDII-GLuc (NF-κB reporter) cells expressing UL37-V5 WT or E1101A TRAF6-binding mutant (24 hpt). (Bottom) Relative GLuc in S/N from 293T PRDII-GLuc cells expressing UL37-V5 WT or E1101A. (H) Immunoblot analysis of 293T MAVSKO IFNB1-GLuc cells following expression of Flag-MAVS as well as UL37-V5 WT and E1101A at 36 hpt. (I) Quantification of phosphorylated IRF3 (S386) relative to total IRF3 from experiments in (H). (J) Relative GLuc in the S/N from 293T MAVSKO IFNB1-GLuc cells following overexpression of Flag-MAVS and UL37-V5 WT and E1101A (36 hpt). (K) RT-qPCR analysis of IFNB1 (target of IRF3 and NF-κB), IFIT1 (target of IRF3 and ISGF3 after activation by interferons), and IL8 (target of NF-κB) mRNA expression relative to HPRT1 from 293T MAVSKO IFNB1-GLuc cells following overexpression of Flag-MAVS and UL37-V5 WT and E1101A (36 hpt).

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-TRAF6 (CST, 1:1000), rabbit anti-IRF3 (CST, 1:1000), rabbit anti-IRF3 phospho-S386 (Abcam, 1:1000), HRP-conjugated anti-FLAG (Sigma-Aldrich, 1:5000), HRP-conjugated anti-V5 (Proteintech, 1:5000) and HRP-conjugated anti-β-actin (CST, 1:5000).

Techniques: Virus, Binding Assay, Luciferase, Over Expression, Transfection, Western Blot, Mutagenesis, Immunoprecipitation, Expressing, Quantitative RT-PCR, Activation Assay