Journal: iScience

Article Title: ZFAND6 promotes TRAF2-dependent mitophagy to restrain cGAS-STING signaling

doi: 10.1016/j.isci.2024.111544

Figure Lengend Snippet: Impaired mitophagy in Zfand6 −/− BMDMs (A) Confocal microscopy with fluorescently conjugated antibodies to the lysosomal marker LAMP1 (cyan) and the mitochondrial marker HSP60 (magenta) in WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A in the presence of 100 μM leupeptin. Scale bars represent 5 μm. (B) Confocal microscopy with primary antibody to LC3 and fluorescently conjugated secondary antibody in WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 h in the presence of 20 nM bafilomycin A1, and incubated with 200 nM MitoTracker Deep Red. Each data point represents the number of LC3 foci in a cell. Scale bars represent 5 μm. (C) Western blotting of lipidated LC3 (LC3-II) in WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 h in the presence of 20 nM bafilomycin A1. (D) Transmission electron microscopy of WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A in the presence of 20 nM bafilomycin A1. Scale bars represent 1 μm. (E) Western blotting of the indicated proteins from WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 18 h. Densitometric analysis was performed for TRAF2, HSP60 and MTCO2 using Image Lab. The number reported for each protein represents the relative fold change compared to WT 0 h and normalized to vinculin. Each data point represents an independent biological replicate. Unpaired Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns = not significant.

Article Snippet: Rabbit polyclonal TRAF2 , Proteintech , Cat#26846-1-AP; RRID: AB_2880656.

Techniques: Confocal Microscopy, Marker, Incubation, Western Blot, Transmission Assay, Electron Microscopy

Journal: iScience

Article Title: ZFAND6 promotes TRAF2-dependent mitophagy to restrain cGAS-STING signaling

doi: 10.1016/j.isci.2024.111544

Figure Lengend Snippet: ZFAND6 is required for TRAF2-cIAP1 interactions in response to acute mitochondrial damage (A and B) CoIP assays were performed with lysates from 293T cells transfected with the indicated plasmids. Immunoprecipitations and western blotting were performed with the indicated antibodies to examine ZFAND6-TRAF2 (A) and ZFAND6-cIAP1 (B) interactions. (C) CoIP assay was performed with lysates from WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 h in the presence of 100 μM leupeptin. IPs were performed with either isotype control antibody or TRAF2 antibody. Western blotting was performed with the indicated antibodies. (D) Confocal microscopy was performed to assess TRAF2 translocation to the mitochondria stained with 200 nM MitoTracker Deep Red using primary antibody specific for TRAF2 and fluorescently conjugated secondary antibody in WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 6 h in the presence of 100 μM leupeptin. Each data point represents Mander’s overlap (M2) of TRAF2 translocation to mitochondria. Scale bars represent 5 μm. (E and F) Confocal microscopy was performed with antibodies to total ubiquitin or phospho-ubiquitin (Ser65) in WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 h in the presence of 100 μM leupeptin. Each data point represents an average number of foci/cell from approximately 10 cells for total ubiquitin. For phospho-ubiquitin, each data point represents one cell. Scale bars represent 5 μm. (G) Western blotting of phospho-ubiquitin (Ser65) from WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 h in the presence of 100 μM leupeptin. Unpaired Student’s t test. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns = not significant.

Article Snippet: Rabbit polyclonal TRAF2 , Proteintech , Cat#26846-1-AP; RRID: AB_2880656.

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Control, Confocal Microscopy, Translocation Assay, Staining, Ubiquitin Proteomics

Journal: iScience

Article Title: ZFAND6 promotes TRAF2-dependent mitophagy to restrain cGAS-STING signaling

doi: 10.1016/j.isci.2024.111544

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal TRAF2 , Proteintech , Cat#26846-1-AP; RRID: AB_2880656.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Reverse Transcription, Blocking Assay, SYBR Green Assay, DNA Extraction, Enzyme-linked Immunosorbent Assay, Mutagenesis, RNA Sequencing, Sequencing, Software, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Identification of Overexpressed Genes in Malignant Pleural Mesothelioma

doi: 10.3390/ijms22052738

Figure Lengend Snippet: Multivariate Cox analysis of the three gene signatures: ( A ) unreduced; ( B ) FDR-reduced; ( C ) GO-reduced.

Article Snippet: The membranes were blocked with 5% milk TBST and probed overnight at 4 °C with the specific primary antibody: anti-CIT rabbit polyclonal antibody (1:500; Proteintech, Rosemont, IL, USA); anti-CTHRC1 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-E selectin rabbit polyclonal antibody (1:750; Proteintech, Rosemont, IL, USA); anti-Midkine (MDK) rabbit polyclonal antibody (1:500; Proteintech, Rosemont, IL, USA); anti-SPARC rabbit polyclonal antibody (1:750; Proteintech, Rosemont, IL, USA); anti-TRAF2 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-UHRF1 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-DSC3 mouse polyclonal antibody (1:500; Genetex, Irvine, CA, USA); anti-KIF23 rabbit polyclonal antibody (1:500; OriGene, Rockville, MD, USA); anti-PRSS23 rabbit polyclonal antibody (1:500; Abcam, Cambridge, MA, USA); anti-ADAMTS1 rabbit polyclonal antibody (1:500; GeneTex, Irvine, CA, USA); anti-PODXL rabbit polyclonal antibody (1:500; Proteintech, Rosemont, IL, USA); anti-BAG2 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-TNNT1 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-MAD2L1 rabbit polyclonal antibody (1:800; Proteintech, Rosemont, IL, USA).

Techniques:

Journal: OncoTargets and therapy

Article Title: RIG-I Promotes Cell Death in Hepatocellular Carcinoma by Inducing M1 Polarization of Perineal Macrophages Through the RIG-I/MAVS/NF-κB Pathway

doi: 10.2147/OTT.S258450

Figure Lengend Snippet: Overexpression of RIG-I activates NF-κB pathway in macrophages. ( A ) Peritoneal macrophages were infected with RIG-I lentivirus or negative control lentivirus and subsequently treated with 100 ng/mL LPS and 20 ng/mL IFN-γ for 24 hours. The levels of RIG-I, MAVS, TRAF2, TRAF3, p65 and IκBα were detected by Western blotting, as well as the phosphorylated p65 and IκBα. ( B ) The gray value of each band was analyzed with Image J software. The expression of RIG-I, MAVS, TRAF2, TRAF3, p65 and IκBα were shown in histogram. **p<0.01, compared with control group.

Article Snippet: Anti-TRAF2 Homo sapiens TNF receptor-associated factor 2 (TRAF2) (cat. no. TA319201) is a rabbit polyclonal antibody and was purchased from OriGene (Beijing, China), and the dilution was 1 μg/mL.

Techniques: Over Expression, Infection, Negative Control, Western Blot, Software, Expressing, Control

Journal: OncoTargets and therapy

Article Title: RIG-I Promotes Cell Death in Hepatocellular Carcinoma by Inducing M1 Polarization of Perineal Macrophages Through the RIG-I/MAVS/NF-κB Pathway

doi: 10.2147/OTT.S258450

Figure Lengend Snippet: Overexpression of RIG-I activates NF-κB pathway in macrophages. ( A ) Peritoneal macrophages were infected with RIG-I lentivirus or negative control lentivirus and subsequently treated with 100 ng/mL LPS and 20 ng/mL IFN-γ for 24 hours. The levels of RIG-I, MAVS, TRAF2, TRAF3, p65 and IκBα were detected by Western blotting, as well as the phosphorylated p65 and IκBα. ( B ) The gray value of each band was analyzed with Image J software. The expression of RIG-I, MAVS, TRAF2, TRAF3, p65 and IκBα were shown in histogram. **p<0.01, compared with control group.

Article Snippet: Anti-TRAF2 Homo sapiens TNF receptor-associated factor 2 (TRAF2) (cat. no. TA319201) is a rabbit polyclonal antibody and was purchased from OriGene (Beijing, China), and the dilution was 1 μg/mL.

Techniques: Over Expression, Infection, Negative Control, Western Blot, Software, Expressing