Journal: Scientific Reports

Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

doi: 10.1038/srep25353

Figure Lengend Snippet: ( a ) The cartoon diagrammatic drawing shows the processes of screening. ( b ) Typical fluorescence microscopy images verify the PpIX binding specificity of the protein expressed on selected phage clone. Selected phage clone shows significant PpIX binding (red) compared to no-PpIX-added and non-specific phage clones. eEF1A1 antibody blockade eliminate the PpIX binding. Scale bar is 10 μm. ( c ) The PpIX MFI of n = 20 phage clones in each data set. Mean ± SD.

Article Snippet: The cells were permeabilized by 0.5% Triton X-100 (Sigma) for 1 h at RT and treated with blocking buffer containing 2% BSA, 10% goat serum (Gibco) and 0.1% Tween-200 (Sigma) for 1 h. The samples were incubated with rabbit anti-human eEF1A1 polyclonal antibody (10 μg/ml) at 4 °C overnight and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (10 μg/ml, Boster Biotechnology Corp., Wuhan, China) at 37 °C for 30 min. All the operations were protected from light.

Techniques: Fluorescence, Microscopy, Binding Assay, Clone Assay

Journal: Scientific Reports

Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

doi: 10.1038/srep25353

Figure Lengend Snippet: ( a ) Correlations of coated purified eEF1A1 protein and BSA (control) or binding PpIX (fluorescence intensity). n = 4 samples per data set; Mean ± SD. ( b ) The representative images present the co-localization of FITC-labeled eEF1A1 (green) and PpIX (red) in situ acquired by both CLSM and SIM. Areas highlighted by white dot frames in whole cell images are zoomed to show details. Scale bars for whole cell images are 10 μm. Scale bar for zoomed CLSM image is 3 μm. Scale bars for zoomed SIM images are 1 μm.

Article Snippet: The cells were permeabilized by 0.5% Triton X-100 (Sigma) for 1 h at RT and treated with blocking buffer containing 2% BSA, 10% goat serum (Gibco) and 0.1% Tween-200 (Sigma) for 1 h. The samples were incubated with rabbit anti-human eEF1A1 polyclonal antibody (10 μg/ml) at 4 °C overnight and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (10 μg/ml, Boster Biotechnology Corp., Wuhan, China) at 37 °C for 30 min. All the operations were protected from light.

Techniques: Purification, Control, Binding Assay, Fluorescence, Labeling, In Situ

Journal: Scientific Reports

Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

doi: 10.1038/srep25353

Figure Lengend Snippet: ( a–d ) The expression of eEF1A1 in HepG2 and L02 cells ( a , c ) or HepG2 cells transfected by eEF1A1 reconstructed or control plasmids ( b , d ) assessed by western blot ( a , b ) and qRT-PCR ( c , d ). ( e,f ) Time-lapse measurements of ALA-induced PpIX accumulations in HepG2 and L02 cells ( e ) or HepG2 cells transfected by eEF1A1 reconstructed or control plasmids ( f ). Mean ± SD, n = 4 samples per data set, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were permeabilized by 0.5% Triton X-100 (Sigma) for 1 h at RT and treated with blocking buffer containing 2% BSA, 10% goat serum (Gibco) and 0.1% Tween-200 (Sigma) for 1 h. The samples were incubated with rabbit anti-human eEF1A1 polyclonal antibody (10 μg/ml) at 4 °C overnight and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (10 μg/ml, Boster Biotechnology Corp., Wuhan, China) at 37 °C for 30 min. All the operations were protected from light.

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

doi: 10.1038/srep25353

Figure Lengend Snippet: Time-lapse monitoring of PpIX MFI in lysates with or without adding eEF1A1 protein. PpIX is degraded significantly slower when eEF1A1 is presentcompared to that in the control. Mean ± SD, n = 3 samples per data set, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The cells were permeabilized by 0.5% Triton X-100 (Sigma) for 1 h at RT and treated with blocking buffer containing 2% BSA, 10% goat serum (Gibco) and 0.1% Tween-200 (Sigma) for 1 h. The samples were incubated with rabbit anti-human eEF1A1 polyclonal antibody (10 μg/ml) at 4 °C overnight and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (10 μg/ml, Boster Biotechnology Corp., Wuhan, China) at 37 °C for 30 min. All the operations were protected from light.

Techniques: Control

Journal: Life Science Alliance

Article Title: miR-486 is essential for muscle function and suppresses a dystrophic transcriptome

doi: 10.26508/lsa.202101215

Figure Lengend Snippet: (A) Schematic demonstrating the workflow for the chimeric eCLIP sequencing platform to identify miR-486 in vivo skeletal muscle regulated transcripts. Tibialis anterior muscles were harvested from 6-mo-old WT and mir-486 KO male mice, and total RNA isolation was completed. The Ago2-miR-486 complex bound to target RNA transcripts was isolated, and then sequencing was performed to map the miR-486–associated peak tracks. The sequence logo (AUGUACAG) represents the consensus sequence for the top 18 miR-486–associated peak reads based on the miRNA:mRNA target sequencing alignments. The miR-486 seed sequence is shown in the 5′–3′ direction. (B) Chromosomal location of a single top hit peak transcript, Mt2 , as identified by eCLIP-seq as a direct target of miR-486. Peaks generated using Integrative Genome Viewer. (C) Metagene plot demonstrating overall miR-486 binding location by a relative position on the target gene. (D) Pie chart demonstrating the proportion of miR-486 gene targets and the respective intragenic binding location of miR-486. (E) Table outlining the top 10 transcripts that were identified as direct targets of miR-486 via CLIP-seq. (F) g:Profiler enrichment analysis graph demonstrates the most significant cellular pathways associated with the 18 direct miR-486 targets identified via chimeric eCLIP-seq. The pathway ID number in the table correlates with the numbered dots in the accompanying graph above. (G) Heat map expression of quantitative PCR of 18 miR-486 eCLIP-seq targets in mir-486 KO and mdx 5cv tibialis anterior muscles expression levels compared with WT controls in separate cohort analyses. Data points are individual biological replicates, N = 4/cohort, and logarithmic fold change normalized to both wild type and β-actin is shown. ** P ≤ 0.01 N = 3 replicates per cohort. Transcript levels are normalized to β-actin, and mir-486 KO levels are shown as relative to WT.

Article Snippet: A total of 10% of IP samples and 1% of input samples were run on NuPAGE 4–12% Bis-Tris protein gels, transferred to PVDF membrane, and probed with 1:1,000 of AGO2 WB antibody and 1:10,000 TrueBlot anti-Rabbit IgG (HRP) (Rockland Immunochemicals) and imaged with a C300 Imager for 1 min on normal settings using Azure Radiance ECL.

Techniques: Sequencing, In Vivo, Muscles, Isolation, Generated, Binding Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes.

doi: 10.4049/jimmunol.180.7.5067

Figure Lengend Snippet: FIGURE 1. Binding of HMGB1 to LPS. A, Six His-tagged wild type HMGB1 and C-HMGB1 proteins were expressed in E. coli and purified. Each protein was observed at the expected size by Coomassie blue staining after SDS-PAGE. B and D, Microtiter plates were immobilized with 10 g/ml S. minnesota LPS and the binding of each His-tagged HMGB1, C-HMGB1 protein, and HMGB1 boxes A and B were tested by ELISA. GST protein was used as a negative control. C, Microtiter plate was im- mobilized with 5 g/ml HMGB1 and variable concentrations of biotin-LPS were incubated to test the binding to HMGB1.

Article Snippet: Eukaryotic recombinant human HMGB1 (R&D Systems), which was expressed in a mammalian cell of mouse myeloma cell line NS0, was used to confirm this assay.

Techniques: Binding Assay, Staining, SDS Page, Enzyme-linked Immunosorbent Assay, Negative Control, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes.

doi: 10.4049/jimmunol.180.7.5067

Figure Lengend Snippet: FIGURE 2. Competitive binding of HMGB1 and LBP to LPS. Micro- titer plates were immobilized with 1 g/ml S. minnesota LPS and a con- stant amount of each His-tagged HMGB1 protein was added to the wells in the presence of various concentrations of LBP. The binding of HMGB1 to LPS was measured by ELISA. PMB was used as a positive control.

Article Snippet: Eukaryotic recombinant human HMGB1 (R&D Systems), which was expressed in a mammalian cell of mouse myeloma cell line NS0, was used to confirm this assay.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes.

doi: 10.4049/jimmunol.180.7.5067

Figure Lengend Snippet: FIGURE 3. Surface plasmon resonance analyses of LPS binding to HMGB1. Three types of LPS isolated from S. minnesota, S. typhi, E. coli 0111:B4, and two types of mutant LPS of Re mutant LPS from S. minnesota Re595 and of delipidated LPS from E. coli 0111:B4, were flowed over HMGB1-, C-HMGB1-, and HMGB1 boxes A and B-immobilized CM5 dextran sensor chips. Three types of wild-type LPS were applied at 0.63, 1.25, 2.5, 5, and 10 M, and Re mutant LPS and delipidated LPS were applied at 0.06, 0.13, 0.25, 0.5, and 1 g/ml. An activated and blocked flow-cell without immobilized ligand was used to evaluate nonspecific binding. n.d., Not done.

Article Snippet: Eukaryotic recombinant human HMGB1 (R&D Systems), which was expressed in a mammalian cell of mouse myeloma cell line NS0, was used to confirm this assay.

Techniques: SPR Assay, Binding Assay, Isolation, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes.

doi: 10.4049/jimmunol.180.7.5067

Figure Lengend Snippet: FIGURE 4. Native gel mobility shift assay of LPS transfer to sCD14 by HMGB1. A, E. coli LPS or S. minnesota LPS was incubated with HMGB1 for 1.5 h at 37°C and subjected to 4–20% native PAGE for membrane transfer. The membrane was blotted with anti-HMGB1 and reblotted with anti-LPS. B, One g/ml S. minnesota LPS was incubated with the indicated amount of HMGB1 or LBP in the presence of sCD14 and subjected to native PAGE. The membrane was blotted with anti-CD14.

Article Snippet: Eukaryotic recombinant human HMGB1 (R&D Systems), which was expressed in a mammalian cell of mouse myeloma cell line NS0, was used to confirm this assay.

Techniques: Mobility Shift, Incubation, Clear Native PAGE, Membrane

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes.

doi: 10.4049/jimmunol.180.7.5067

Figure Lengend Snippet: FIGURE 5. HMGB1-mediated transfer of BODIPY FL-LPS to sCD14 protein and human PBMCs. A and B, A mixture of BODIPY FL-LPS and sCD14 was incubated in the presence of bacterially produced HMGB1 (Bact. HMGB1; A) or eukaryotic recombinant HMGB1 (Euk. HMGB1; B), and fluorescence levels were measured after 10 h at 25°C. LBP was used as a positive control and 2% SDS was used to completely solubilize the disaggregated state of LPS for maximum fluorescence. Heat-treated (tx) HMGB1 was added to confirm the effect of HMGB1. Each fluorescence level is shown as the mean SD of three experiments. , p 0.001, vs the treatment of BODIPY-LPS with sCD14. C, The real-time change in BODIPY FL-LPS fluorescence levels was recorded for 240 min at 5-min intervals after incubation. A mixture of BODIPY FL-LPS (1 g/ml) and sCD14 (5 g/ml) was incubated with bacterially produced recombinant HMGB1 (5 g/ml) or LBP (2 g/ml) or none as described in Materials and Methods. D, Human PBMCs of 5 105 cells were incubated with a mixture of BODIPY-LPS and HMGB1 or LBP for 60 min at 37°C. The mixture was preincubated for 60 min at 37°C before its addition to the cells. The cell-associated fluorescence levels were measured after washing. The figure is a representative of two independent experiments with similar results.

Article Snippet: Eukaryotic recombinant human HMGB1 (R&D Systems), which was expressed in a mammalian cell of mouse myeloma cell line NS0, was used to confirm this assay.

Techniques: Incubation, Produced, Recombinant, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes.

doi: 10.4049/jimmunol.180.7.5067

Figure Lengend Snippet: FIGURE 6. TNF- production in human PBMCs and PBMo after HMGB1-mediated LPS transfer. A and B, Human PBMCs (5 106 cells/ ml; A) and PBMo (5 105 cells/ml; B) were stimulated with the indicated concentrations of LPS in the presence of HMGB1 or LBP for 16 h at 37°C. The figures are representatives of three and two independent experiments with similar results, respectively. Heat-treated (tx) HMGB1 was added to confirm the effect of HMGB1. PMB was added at the concentration of 10 g/ml.

Article Snippet: Eukaryotic recombinant human HMGB1 (R&D Systems), which was expressed in a mammalian cell of mouse myeloma cell line NS0, was used to confirm this assay.

Techniques: Concentration Assay

Journal: EMBO Reports

Article Title: Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase

doi: 10.1038/s44319-025-00397-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-eIF4E (Boster) was used at 1:10000.

Techniques: SYBR Green Assay, Imaging, Recombinant, Sequencing, Software, Microscopy, Ion-Mobility Spectrometry, Mass Spectrometry