Journal: Cell Death & Disease

Article Title: TRPV1 alleviates osteoarthritis by inhibiting M1 macrophage polarization via Ca 2+ /CaMKII/Nrf2 signaling pathway

doi: 10.1038/s41419-021-03792-8

Figure Lengend Snippet: A Western blot and B quantitative analysis of cytosolic and nuclear Nrf2 protein levels in cells treated with LPS and LPS + CPS with or without ML385 for 3 h. C Immunofluorescence image of Nrf2 and D its quantitative analysis in RAW264.7 cells treated with LPS and LPS + CPS with or without ML385 for 3 h. White arrow: cells with Nrf2 nuclear translocation. E qPCR analysis of mRNA levels of M1 macrophage markers in RAW264.7 cells treated with LPS and LPS + CPS with or without ML385 for 24 h. F Western blot and quantitative analysis of iNOS and COX2 protein expression levels in RAW264.7 cells treated with LPS and LPS + CPS with or without ML385 for 48 h. Data ( n = 3) are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bars: 10 μm. NS nonsignificant.

Article Snippet: In the cytological immunofluorescence, the cells were fixed in 4% PFA and permeated by 0.1% Triton X-100 for 15 min. After blocking with 5% BSA, the slides were incubated with primary antibody for Nrf2 (#16396-1-AP, 1:200, Proteintech).

Techniques: Western Blot, Immunofluorescence, Translocation Assay, Expressing

Journal: Cell Death & Disease

Article Title: TRPV1 alleviates osteoarthritis by inhibiting M1 macrophage polarization via Ca 2+ /CaMKII/Nrf2 signaling pathway

doi: 10.1038/s41419-021-03792-8

Figure Lengend Snippet: A Fluorescence images and B their quantitative analysis of intensity in the Fluo-4 AM-loaded RAW264.7 cells treated with CaCl 2 , CPS, or CaCl 2 + CPS under the Ca 2+ -free medium for 10, 30, 60, and 90 min. Scale bars: 50 μm. C Western blot analysis of cytosolic and nuclear Nrf2 protein levels in the cells treated with LPS, LPS + CPS, and LPS + CPS with EDTA (left) or Ca 2+ -free medium (right) for 3 h. D Quantitative analysis and E representative images of Nrf2 immunofluorescence in the cells treated with LPS, LPS + CPS, and LPS + CPS with EDTA or Ca 2+ -free medium for 3 h. Scale bars: 10 μm. White arrow: cells with Nrf2 nuclear translocation. Data ( n = 3) are shown as mean ± SD. * P < 0.05; *** P < 0.001; NS nonsignificant.

Article Snippet: In the cytological immunofluorescence, the cells were fixed in 4% PFA and permeated by 0.1% Triton X-100 for 15 min. After blocking with 5% BSA, the slides were incubated with primary antibody for Nrf2 (#16396-1-AP, 1:200, Proteintech).

Techniques: Fluorescence, Western Blot, Immunofluorescence, Translocation Assay

Journal: Cell Death & Disease

Article Title: TRPV1 alleviates osteoarthritis by inhibiting M1 macrophage polarization via Ca 2+ /CaMKII/Nrf2 signaling pathway

doi: 10.1038/s41419-021-03792-8

Figure Lengend Snippet: A Western blot and quantitative analyses of CaMKII and p-CaMKII protein levels in the cells treated with LPS with or without CPS for 5, 15, and 30 min. B Western blot analysis of cytosolic and nuclear Nrf2 protein levels in the cells treated with LPS and LPS + CPS with or without KN-93 for 3 h. C Immunofluorescence images and D quantitative analysis of Nrf2 in the cells treated with LPS and LPS + CPS with or without KN-93 for 3 h. Scale bars: 10 μm. Data ( n = 3) are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; NS nonsignificant.

Article Snippet: In the cytological immunofluorescence, the cells were fixed in 4% PFA and permeated by 0.1% Triton X-100 for 15 min. After blocking with 5% BSA, the slides were incubated with primary antibody for Nrf2 (#16396-1-AP, 1:200, Proteintech).

Techniques: Western Blot, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Binding of zebrafish lipovitellin and L1-ORF2 increases the accessibility of L1-ORF2 via interference with histone wrapping

doi: 10.3892/ijmm.2024.5443

Figure Lengend Snippet: Primers used for reverse transcription-quantitative PCR.

Article Snippet: Expression levels of the EZE expression genes GRHL3 (grainyhead-like transcription factor 3), SOX19A (SRY-box transcription factor 19a) and NNR (nanor) were detected using qPCR kit (cat. no. QP031, GeneCopoeia). qPCR was performed using the following thermocycling conditions: Initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 10 sec and 56°C for 40 sec.

Techniques: Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Binding of zebrafish lipovitellin and L1-ORF2 increases the accessibility of L1-ORF2 via interference with histone wrapping

doi: 10.3892/ijmm.2024.5443

Figure Lengend Snippet: LV activates EGFP gene expression in good-quality 0 h ZEs and promotes expression both of EGFP reporter gene and genes of EZEs. (A) Embryos injected with C1-delPolyA-ORF2, C1-delPolyA-ZORF2, C1 and C1-delPolyA expression vectors. The scale bar represents 200 μ m. (B) EGFP fluorescence intensity. (C) LV attenuates the inhibitory effect of histone on EGFP mRNA. LV promotes expression of GRHL3, SOX19A and NNR genes in (D) EZEs and (E) adult male zebrafish liver. * P<0.01, ** P<0.01, *** P<0.001, **** P<0.0001. (F) GO gene function classification. GO, Gene Ontology; GRHL3, grainyhead-like transcription factor 3; SOX19A, SRY-box transcription factor 19a; NNR, nanor. BP, biological process; CC, cell component; MF, molecular function; LV, lipovitellin; EGFP, enhanced green fluorescent protein; ORF2, open reading frame 2.

Article Snippet: Expression levels of the EZE expression genes GRHL3 (grainyhead-like transcription factor 3), SOX19A (SRY-box transcription factor 19a) and NNR (nanor) were detected using qPCR kit (cat. no. QP031, GeneCopoeia). qPCR was performed using the following thermocycling conditions: Initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 10 sec and 56°C for 40 sec.

Techniques: Gene Expression, Expressing, Injection, Fluorescence

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 negatively regulated IFN-β induced by H13N2 infection. (a, B) luciferase reporter assays were used to assess the impact of FGF8 overexpression on IFN-β and ISRE promoter activity in A549 cells infected with H13N2 at an MOI of 1. (C-F) FGF8-overexpressing A549 cells were infected with H13N2 at an MOI of 1. At 12 hours post-infection (hpi), IFN-β levels in the cell supernatant were measured using ELISA (C), and IFN-β mRNA levels were evaluated by RT-qPCR (d). At 24 hpi, the mRNA levels of interferon-stimulated genes MX1 (e) and IFIT1 (f) were assessed by RT-qPCR. (G-J) stable FGF8-knockdown A549 cells were infected with H13N2 at an MOI of 1. At 12 hpi, IFN-β levels in the cell supernatant were quantified by ELISA (G), and IFN-β mRNA levels were evaluated using RT-qPCR (H). At 24 hpi, the mRNA levels of MX1 (i) and IFIT1 (J) were assessed by RT-qPCR. (K and L) Western blot analysis evaluated RIG-I, p-TBK1, and p-IRF3 expression in A549 cells with FGF8 overexpression (L) or knockdown (K) at 12 hours after H13N2 infection (MOI = 1). Band intensities were quantified by densitometric analysis. Statistical analysis was performed using two-tailed unpaired Student’s t-tests, with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Infection, Luciferase, Over Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 drives ubiquitin – proteasomal degradation of RIG-I. (a) FGF8 inhibits RIG-I-mediated signaling. A luciferase reporter assay was performed to evaluate the effect of FGF8 overexpression on IFN-β promoter activation induced by RIG-I. (B and C) FGF8 does not affect RIG-I transcription. RIG-I mRNA levels were quantified by RT-qPCR in FGF8-overexpressing A549 cells at 0, 6, and 12 hours post-infection with H13N2 (b) or H1N1 (C) at an MOI of 1. (d) dose-dependent reduction of RIG-I protein. A549 cells were transfected with increasing amounts of Flag-FGF8 plasmid for 24 hours, followed by infection with H13N2 (MOI = 1) for 12 hours. RIG-I protein levels were analyzed by Western blot, and band intensities were quantified by densitometry. (e) FGF8 reduces RIG-I stability. FGF8-overexpressing A549 cells were infected with H13N2 (MOI = 1) and treated with cycloheximide (CHX, 50 µg/mL) for the indicated time periods. Protein levels were analyzed by Western blot, and the relative abundance of HA-RIG-I was quantified to assess protein degradation rates. (F and G) proteasome inhibition restores RIG-I levels. A549 cells infected with H13N2 (f) or H1N1 (G) at an MOI of 1 were treated with DMSO, chloroquine (CQ, 50 µM), or MG132 (10 µM) for 6 hours. RIG-I expression was analyzed by Western blot, with relative protein levels quantified by densitometry. (H and I) FGF8 promotes K48-linked ubiquitination of RIG-I. HEK-293T cells were co-transfected with the indicated plasmids and treated with MG132 for 6 hours. (H) Total ubiquitination of RIG-I was assessed by immunoprecipitation with anti-HA antibody followed by immunoblotting (ib) with anti-Myc. (i) K48- or K63-linked ubiquitination was analyzed using specific ubiquitin mutants. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Luciferase, Reporter Assay, Over Expression, Activation Assay, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Western Blot, Inhibition, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: TRIM16 mediated RIG-I degradation and promoted influenza virus replication. (a) Co-immunoprecipitation analysis was performed in cells transfected with Flag-TRIM16 and HA-RIG-I, with or without H13N2 infection (MOI = 1), to verify the interaction. (b) immunofluorescence microscopy showing the localization of TRIM16 (green) and RIG-I (red) in cells infected with H13N2 or mock-infected (NC). Nuclei were stained with DAPI (blue). Note that TRIM16 and RIG-I show diffuse distribution in the NC group but form co-localized puncta (yellow) upon H13N2 infection. Scale bar: 5 μm. (C) in vitro ubiquitination assay to verify the direct E3 ligase activity of TRIM16 using wt and ΔB-Box mutant proteins. (d) in vitro ubiquitination assay to determine the linkage specificity of TRIM16-mediated RIG-I ubiquitination using K48-only and K63-only ubiquitin mutants. (e) bioinformatic analysis using PONDR revealed the presence of intrinsically disordered regions (IDRs) in the FGF8 protein sequence. (f) fluorescence microscopy of A549 cells transfected with EGFP-FGF8 (green). Nuclei were stained with DAPI. Scale bar represents 10 μm. (G) TurboID-based proximity labeling assay was performed in cells expressing FGF8-TurboID. Biotinylated proteins were captured using streptavidin beads, and the pulled-down proteins were analyzed by Western blot to detect the presence of RIG-I and TRIM16. (H and I) validation of TRIM16 knockdown. RT-qPCR (H) and Western blot (i) confirmed the silencing efficiency in A549 cells. (J) control and TRIM16-silenced A549 cells were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein levels (NP, PB1, PB2) were analyzed by Western blot, and band intensities were quantified by densitometry. (K) RT-qPCR analysis of IFN-β mRNA levels in TRIM16-silenced A549 cells 12 hours post-infection with H13N2 (MOI = 1). (L) Western blot confirmation of TRIM16 overexpression (OE-TRIM16). (M) A549 cells overexpressing TRIM16 were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein expression was analyzed by Western blot and quantified by densitometry. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Virus, Immunoprecipitation, Transfection, Infection, Immunofluorescence, Microscopy, Staining, In Vitro, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Sequencing, Fluorescence, Labeling, Expressing, Western Blot, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Control, Over Expression, Two Tailed Test

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: CUL1 and UBE2L3 overexpression increases the ubiquitination and decreases HPV16 E7 protein level in HPV+ HNC cells. ( A ) The schematic model of HPV16 E7 degradation through CUL1- and UBE2L3-mediated ubiquitination. Figure generated with a Biorender icon. CUL1 or UBE2L3 is overexpressed in HPV+ HNC (SCC152) ( B–C ) and SCC2 ( D–E ) cells using lentiviral transduction. CUL1, UBE2L3, HPV16 E7, and MARCHF8 proteins were detected by western blotting. The relative band intensities were quantified using NIH ImageJ in SCC152 ( C ) and SCC2 ( E ). β-Actin was used as an internal control. ( F ) SCC152 cells with CUL1 or UBE2L3 overexpression were treated with MG132 (10 µM). Ubiquitinated proteins were pulled down from the cell lysate using anti-ubiquitin antibody-conjugated magnetic beads, and HPV16 E7 protein was detected by western blotting. The data shown are means ± SD of three independent experiments. Student’s t -test was used to determine P -values. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Over Expression, Ubiquitin Proteomics, Generated, Transduction, Western Blot, Control, Magnetic Beads

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: CUL1 and UBE2L3 protein levels are low in HPV+ HNC cells. CUL1 and UBE2L3 protein levels in normal (N/Tert-1), HPV- HNC (SCC1, SCC9, and SCC19), and HPV+ HNC (SCC2, SCC90, and SCC152) cells were determined by western blotting ( A ). Relative band intensities were quantified using NIH ImageJ ( B ). HPV16 E7 and β-actin were used as viral and internal controls, respectively. CUL1 and UBE2L3 mRNA expression levels in normal (N/Tert-1), HPV+ HNC (SCC2, SCC90, and SCC152), and HPV− HNC (SCC1, SCC9, and SCC19) cells were quantified by RT-qPCR ( C ). The data shown are normalized by the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level as an internal control. The mRNA expression levels of CUL1 and UBE2L3 were analyzed using our previous gene expression data (GSE6791) and shown as fluorescence intensity (log2) from microdissected human tissue samples from HPV+ ( n = 16) and HPV− ( n = 26) HNC patients and normal individuals ( n = 12) ( D ). CUL1, UBE2L3, and E7 protein levels in SCC152 cells treated with MG132 (10 µM) or dimethyl sulfoxide (DMSO) ( E ). Relative band intensities were quantified using NIH ImageJ ( F ). CUL1 and UBE2L3 proteins were detected in N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 by western blotting ( G ). The size of HPV16 E7 in N/Tert-1 E7 cells is about 22 kDa due to HA tagging, while the size of native HPV16 E7 in N/Tert-1 E6E7 cells is about 17 kDa. Relative band intensities were quantified using NIH ImageJ ( H ). Total RNA was extracted from N/Tert-1 containing an empty vector and N/Tert-1 cells expressing HPV16 E6, E7, or E6 and E7 (E6E7). The HPV16 E6 and E7 mRNA levels were quantified by RT-qPCR ( I ). The data shown are normalized by the GAPDH mRNA level as an internal control. All experiments were repeated at least three times, and the data shown are means ± SD of three independent experiments. Student’s t -test determined P -values. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Control, Gene Expression, Fluorescence, Plasmid Preparation

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: Knockdown of MARCHF8 expression increases CUL1 and UBE2L3 protein levels and decreases HPV16 E7 protein levels in HPV+ HNC cells. SCC152 (A and C) and SCC2 (B and D) cells were transduced with five and three lentiviral shR-MARCHF8, respectively, or shR-scr as a control. MARCHF8, CUL1, UBE2L3, HPV16 E7, and pRb proteins in SCC152 ( A ) and SCC2 ( B ) cells were detected by western blotting. The relative band intensities were quantified using NIH ImageJ ( C and D ). β-Actin was used as an internal control. The mRNA levels of CUL1, UBE2L3, and HPV16 E7 were quantified by RT-qPCR in SCC152 ( E ) and SCC2 ( F ) cells transduced with five and three lentiviral shR-MARCHF8, respectively, or shR-scr as a control. The data shown are normalized by the GAPDH mRNA level as an internal control. The data shown are means ± SD of three independent experiments. Student’s t -test determined P -values. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Knockdown, Expressing, Transduction, Control, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: MARCHF8 protein interacts with and ubiquitinates CUL1 and UBE2L3 proteins. MARCHF8 ( A ), CUL1 ( B ), and UBE2L3 ( C ) were pulled down from the lysate of SCC-152 cells treated with MG132 (10 µM) using anti-MARCHF8 ( A ), anti-CUL1 ( B ), and anti-UBE2L3 ( C ) antibodies, respectively. CUL1, UBE2L3, HPV16 E7, and MARCHF8 were detected from the immunoprecipitated proteins by western blotting. ( D–F ) Ubiquitinated proteins were pulled down from the lysate of SCC152 cells with shR-scr or shRNA against MARCHF8 (shR-MARCHF8 clone 3) treated with MG132 (10 µM) using anti-ubiquitin antibody-conjugated magnetic beads. CUL1 ( D ), UBE2L3 ( E ), and HPV16 E7 ( F ) proteins were detected in the immunoprecipitated proteins by western blotting. All experiments were repeated at least three times. The data shown are means ± SD of three independent experiments. Student’s t -test determined P -values. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Immunoprecipitation, Western Blot, shRNA, Ubiquitin Proteomics, Magnetic Beads

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: Marchf8 knockout restores CUL1 and UBE2L3 protein levels in HPV+ mouse oral cancer cells. CUL1 and UBE2L3 protein levels in mouse normal immortalized (NiMOE), HPV− transformed (HPV− MOE), and HPV+ transformed (mEERL) oral epithelial cells were determined by western blotting ( A ). β-actin was used as an internal control. The relative band intensities were quantified using NIH ImageJ ( B ). mEERL cells were transduced with lentiviruses containing Cas9 and one of two sgRNAs against Marchf8 (sgR- Marchf 8-2 and sgR- Marchf 8-3) or scrambled sgRNA (sgR-scr). MARCHF8, CUL1, UBE2L3, and HPV16 E7 proteins were detected by western blotting ( C ). Relative band intensities were quantified using NIH ImageJ ( D ). The data shown are means ± SD of three independent experiments. All experiments were repeated at least three times, and the data shown are means ± SD. Student’s t -test determined P -values. * P < 0.05, *** P < 0.001.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Knock-Out, Transformation Assay, Western Blot, Control, Transduction

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: CUL1 and UBE2L3 overexpression suppresses HPV+ HNC tumor growth in vivo . mEERL cells overexpressing Cul1 or Ube2l3 were generated by lentiviral transduction of the Cul1 or Ube2l3 genes , respectively, and blasticidin selection. CUL1, UBE2L3, and HPV16 E7 proteins were detected by western blotting ( A ). Relative band intensities were quantified using NIH ImageJ ( B ). β-actin was used as an internal control. The data shown are means ± SD of three independent experiments. Student’s t -test determined P -values. * P < 0.05, ** P < 0.01, *** P < 0.001. mEERL/vector ( C ), mEERL/ Cul1 ( D ), or mEERL/ Ube2l3 ( E ) cells were injected into the rear right flank of C57BL/6J mice ( n = 10 per group). Tumor volume was measured twice a week ( C–F ). Survival rates of mice were analyzed using a Kaplan-Meier estimator ( G ). The time to event was determined for each group, with the event defined as a tumor size larger than 2,000 mm 3 . The data shown are means ± SD. P -values of mice injected with mEERL/ Cul1 and mEERL/ Ube2l3 cells compared with mice injected with mEERL/vector cells were determined for tumor growth ( F ) and survival ( G ) by two-way analysis of variance. Shown are representative of two independent experiments.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Over Expression, In Vivo, Generated, Transduction, Selection, Western Blot, Control, Plasmid Preparation, Injection

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: The schematic model of MARCHF8-mediated HPV16 E7 stabilization by degrading CUL1 and UBE2L3. The HPV16 oncoprotein E6 activates the MARCHF8 promoter activity through the MYC/MAX transcription factor complex and upregulates MARCHF8 in the HPV+ HNC cells . MARCHF8 protein ubiquitinates and degrades CUL1 and UBE2L3 proteins, leading to the prevention of HPV16 E7 protein degradation.

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Activity Assay

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: List of the oligonucleotides ^a

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Sequencing, Cloning

Journal: Journal of Virology

Article Title: The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer

doi: 10.1128/jvi.01726-23

Figure Lengend Snippet: List of the antibodies

Article Snippet: UBE2L3 , Human/Mouse , Proteintech , 14115–1-AP , AB_2210891 , IP-WB.

Techniques: Western Blot, Ubiquitin Proteomics

Journal: Materials Today Bio

Article Title: A precise theranostic nanoplatform amplifies anti-tumor efficacy via copper ionophores and sonodynamic therapy

doi: 10.1016/j.mtbio.2026.102957

Figure Lengend Snippet: The anti-tumor immune mechanism of ICCP NPs. a) Schematic illustration of cuproptosis mechanism. b) Immunofluorescence analysis of DLAT and FDX1 in 4T1 cells following various treatments. c) Schematic diagram illustrating the synergistic induction of ICD by SDT and cuproptosis. d) Immunofluorescence analysis of CRT and HMGB1 in 4T1 cells following various treatments. e) Quantitative analysis of CRT fluorescence intensity. f) ATP content in 4T1 cells following various treatments.

Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit polyclonal antibodies against DLAT (13426-1-AP, 1:200), FDX1 (12592-1-AP, 1:250), CRT (10292-1-AP, 1:200), and HMGB1 (10829-1-AP, 1:200) (Proteintech, Wuhan, China).

Techniques: Immunofluorescence, Fluorescence

Journal: Materials Today Bio

Article Title: A precise theranostic nanoplatform amplifies anti-tumor efficacy via copper ionophores and sonodynamic therapy

doi: 10.1016/j.mtbio.2026.102957

Figure Lengend Snippet: The anti-tumor effect of ICCP NPs in vivo . a) Biodistribution of free IR808 and ICCP NPs in tumor-bearing mice. b) NIR imaging of harvested organs 48 h post-injection. c) Schematic diagram of the anti-tumor experiment in BALB/c mice. d) Body weight fluctuations in tumor-bearing mice following various treatments throughout the therapeutic period. e) Tumor volume growth curves in tumor-bearing mice from different treatments. f) Tumor histological assessment by H&E staining, TUNEL, Ki67 staining, and DLAT immunofluorescence following various treatments.

Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit polyclonal antibodies against DLAT (13426-1-AP, 1:200), FDX1 (12592-1-AP, 1:250), CRT (10292-1-AP, 1:200), and HMGB1 (10829-1-AP, 1:200) (Proteintech, Wuhan, China).

Techniques: In Vivo, Imaging, Injection, Staining, TUNEL Assay, Immunofluorescence

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: List of recombinant proteins used in assay development

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques: Recombinant, Expressing, Purification

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: Sensitivity and specificity estimates of salivary and serum assays with 95% confidence intervals

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques:

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: Random forest ranking of proteins included in the final salivary IgG assay

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques: Binding Assay

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: Antibody responses to a parsimonious set of SARS-CoV-2 proteins by COVID-19 status and symptom severity. (A) Spike protein (EPA_S); (B) receptor binding domain (EPA_RBD); (C) nucleocapsid protein (AB_N); (D) stalk subunit S2 of S protein (PS_S2i). Protein IDs are defined in Results and Table 2 . MFI, mean fluorescence intensity.

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques: Binding Assay, Fluorescence

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: Associations between salivary IgG responses to the best-performing SARS-CoV-2 spike protein (EPA_S) and to nucleocapsid protein (AB_N) (A), receptor binding domain (EPA_RBD) (B), and stalk subunit S2 of spike protein (PS_S2i) (C) by COVID-19 status and symptom severity. Seropositivity cutoff line from a logistic regression model with two predictors is shown for the EPA_S-versus-AB_N plot in panel A.

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques: Binding Assay

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: Multiplicative effects with 95% confidence intervals of disease severity categories and age on median salivary antibody responses to SARS-CoV-2 proteins from generalized additive models, adjusted for a spline function of interval from the onset

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques: Binding Assay

Journal: Microbiology Spectrum

Article Title: A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail

doi: 10.1128/Spectrum.00693-21

Figure Lengend Snippet: Time interval from the onset of illness or diagnosis of SARS-CoV-2 infection versus the intensity of salivary IgG responses to recombinant SARS-CoV-2 proteins with a spline function and 95% confidence intervals (dashed lines) for the mean values (solid lines). (A) Spike protein (EPA_S); (B) receptor binding domain (EPA_RBD); (C) nucleocapsid protein (AB_N); (D) stalk subunit of spike protein (PS_S2i).

Article Snippet: The results also demonstrated that the S protein produced at EPA utilizing a plasmid obtained from BEI Resources (EPA_S) was the best-performing protein (ranking top in all 500 runs) while Acro Biosystems (Newark, DE, USA) S and N proteins (AB_S and AB_N, respectively), RBD protein expressed at EPA using another plasmid from BEI Resources (EPA_RBD), and ProSci (Fort Collins, CO, USA) S2 protein (PS_S2i) were among the top five best-performing SARS-CoV-2 proteins ( ).

Techniques: Infection, Recombinant, Binding Assay

Journal: Immunity

Article Title: A CD4 + T cell-intrinsic complement C5aR2-prostacyclin-IL-1R2 axis orchestrates Th1 cell contraction

doi: 10.1016/j.immuni.2025.05.003

Figure Lengend Snippet: (A) Schematic of CD46- and C5aR1/2-controlled human Th1 cell induction (IFN-γ) and contraction (IL-10 co-induction) via modulation of CD4 + T cell-intrinsic IL-1β secretion. (B) Pathway analysis using differentially-expressed genes (DEGs) of human CD4 + T cells activated with CD3 + CD46 (6 h) with or without addition of a C5aR2 antagonist (C5aRA) ( n = 3 donors, one experiment), and prostaglandin pathway is indicated in red and insert. (C) Heatmap depicting the top 10 DEGs derived from (B). (D) IFN-γ and IL-10 production and IFN-γ:IL-10 production ratio of CD3 + CD46 activated (36 h) T cells with or without concurrent incomplete COX-2 inhibition (5 μM) ( n = 8, three independent experiments). (E) Schematic of major prostanoid synthesis enzymes and products downstream of COX-2-generated PGH2, including relevant prostanoid receptors. (F) Kinetics of PTGES2 , TBXAS1 , and PTGIS gene transcription in CD3 + CD46-activated CD4 + T cells ( n = 4, two independent experiments). (G) IFN-γ and IL-10 production and IFN-γ:IL-10 production ratio of CD3 + CD46 activated (36 h) T cells with or without concurrent addition of either PGE2 (left) or prostacyclin (PGI2, right) at 12 h post activation ( n = 4–6, four independent experiments). (H) Intrinsic PGE2 and PGI2 production by non-activated (NA) or subsorted IFN-γ + , IFN-γ + IL-10 + , or IL-10 + T cells after CD3 + CD46 stimulation for either 2 or 36 h ( n = 3–4, two independent experiments). 2 h stimulation generates IFN-γ + cells only; thus, IFN-γ + IL-10 + or IL-10 + cells could not be assessed (na) for prostanoid production at this time point. (I) IFN-γ and IL-10 production by CD3 + CD46 activated (36 h) T cells in which PTGES2 , PTGIS , or PTGIR expression had been inhibited by siRNA treatment as indicated or which had been activated in the presence or absence of an EP4 inhibitor (20 μM) ( n = 3, three independent experiments). (J) Effects of 15-PGDH inhibition with or without concurrent EP4 or PGI2-R antagonism on Th1 cell IFN-γ and/or IL-10 (co)producing cells at 36 h post CD3 + CD46 activation. Representative fluorescence-activated cell sorting (FACS) plots shown on the left (statistical analyses are summarized in ) ( n = 3–4, three independent experiments). (K) Schematic of C5aR2-controlled intrinsic PGE2 vs. PGI2 generation balance and subsequent effects on IFN-γ and IL-10 production by activated Th1 cells. Groups were compared using one-way ANOVA test or paired Student’s t test and error bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01. See also .

Article Snippet: Human TaqMan probe PTGES2 , Thermo Fisher Scientific , CAT# Hs00228159_m1.

Techniques: Derivative Assay, Inhibition, Generated, Activation Assay, Expressing, Fluorescence, FACS

Journal: Immunity

Article Title: A CD4 + T cell-intrinsic complement C5aR2-prostacyclin-IL-1R2 axis orchestrates Th1 cell contraction

doi: 10.1016/j.immuni.2025.05.003

Figure Lengend Snippet: (A) Top increased (orange) and reduced (blue) biological pathways based on differentially expressed genes (DEGs) derived from in vitro CD3 + CD28 stimulated CD4 + T cells isolated from wild-type (WT) or Ptgir −/− (KO) mice ( n = 4, one experiment). (B) Quantification of activated PKA (phosphorylated [pPKA], normalized to total PKA [tPKA]) in IFN-γ + IL-10 − , IFN-γ + IL-10 + , and IFN-γ − IL-10 + CD4 + T cells generated by CD3 + CD46 activation (36 h) ( n = 3, three independent experiments). (C) Amounts of pPKA in CD3 + CD46-activated T cells (18 h) (left) with or without addition of either PGE2 or PGI2 (right) ( n = 5, three independent experiments). (D) Impact of PGE2 vs. PGI2 provision in the presence or absence of a PKA inhibitor (inh.) on IFN-γ and IL-10 production in CD3 + CD46-activated T cells (36 h). Representative FACS plots from one of three donors (left) with statistical analysis of cumulative data (right, n = 3, three independent experiments). (E) Effect of PKA inhibition on IL-1R2 expression by CD3 + CD46-activated T cells (36 h). Representative FACS plots from one of three donors (left) and statistical analysis of cumulative data (right, n = 3, two independent experiments). (F) Amounts of mRNAs encoding PTGES2 , IL1R2 , or IL10 in CD3 + CD46-activated T cells (8 h) with or without PGI2 addition to media assessed by quantitative PCR ( n = 3, one experiment). (G) Effect of PGI2-R expression reduction by PTGIR siRNA treatment in CD3 + CD46-activated T cells (see also ) on IL-10 expression and PKA and CREB activation ( n = 3, three independent experiments). (H) Effect of PGI2-R expression reduction by PTGIR siRNA treatment in CD3 + CD46-activated T cells (48 h) on PTGES2 , IL1R2 , or IL10 transcription ( n = 4–5, four independent experiments). (I) Schematic of the effects of PGE2-EP4 vs. PGI2-PGI2-R activity on CD4 + T cell PKA activation and transcription of indicated genes. The effect of PGI2-R engagement on IL10 transcription vs. translation is not clear (denoted by “?”). Groups were compared using one-way ANOVA or the Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001. See also .

Article Snippet: Human TaqMan probe PTGES2 , Thermo Fisher Scientific , CAT# Hs00228159_m1.

Techniques: Derivative Assay, In Vitro, Isolation, Generated, Activation Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Immunity

Article Title: A CD4 + T cell-intrinsic complement C5aR2-prostacyclin-IL-1R2 axis orchestrates Th1 cell contraction

doi: 10.1016/j.immuni.2025.05.003

Figure Lengend Snippet: (A) IL-1β, IFN-γ, and IL-10 secreted by CD4 + T cells isolated from the indicated individuals with cryopyrin-associated periodic syndrome (CAPS) or sex- and age-matched healthy control donors (HDs) measured at 12–24 h post CD3 + CD46 activation ( n = 5, five independent experiments). (B) Representative FACS plot of cell staining for IFN-γ and IL-10 production by HD3 and CAPS3 CD4 + T cells upon CD3 + CD46 activation (24 h) (of a total of n = 3, three independent experiments). (C) Expression of COX-2, PTGES2, and PTGIS in CD4 + T cells isolated from the indicated individuals with CAPS or HDs measured at 36 h post CD3 + CD46 activation ( n = 3–4, three to four independent experiments). (D) Protein expression of C5aR2, PTGI2-R, and IL-1R2 in CD4 + T cells isolated from the indicated individuals with CAPS or HDs measured at 36 h post CD3 + CD46 activation ( n = 3–4, three to four independent experiments). (E) IL-10 production by T cells isolated from indicated individuals with CAPS after CD3 + CD46 activation with or without either a specific PTGES2 inhibitor (MF63, 1 μM) or soluble IL-1R2 (5 μg/mL) at 12 h post activation ( n = 6–7, six independent experiments). (F) Schematic detailing the shifted balance of the intrinsic prostanoid generation and response machinery toward PGE2 production underlying faulty IL-10 production in CAPS T cells, which could be rescued by PTGES2 inhibition or IL-1R2 provision. Groups were compared using the paired or non-paired Student’s t test, error bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01. See also .

Article Snippet: Human TaqMan probe PTGES2 , Thermo Fisher Scientific , CAT# Hs00228159_m1.

Techniques: Isolation, Control, Activation Assay, Staining, Expressing, Inhibition