Journal: Journal of Biological Chemistry

Article Title: Subcellular Localization of the Aryl Hydrocarbon Receptor Is Modulated by the Immunophilin Homolog Hepatitis B Virus X-associated Protein 2

doi: 10.1074/jbc.m006873200

Figure Lengend Snippet: FIG. 5. Examination of AhR localization and AhR/XAP2 stoichiometry in Hepa-1 cells. A, laser scanning confocal micrographs of AhR in control and 1 nM TCDD-treated Hepa-1 cells visualized with anti-AhR MAb RPT9 and goat anti-mouse IgG conjugated to lissamine-rhodamine sulfonyl chloride, Me2SO (i), 30 min (ii), 1 h (iii), 2 h (iv). B, relative amount of AhR in Hepa-1 cytosolic fraction compared with total AhR (cytosolic 1 nuclear). Cells were treated with 1 nM TCDD for 2 h, harvested, homogenized, and centrifuged to obtain nuclear pellet and crude cytosol (respun to obtain cytoplasm). Nuclear pellet was extracted with MENG (1500 mM NaCl), and soluble extract collected. Aliquots of cytosolic and nuclear extracts were resolved by TSDS-PAGE, blotted to PVDF membrane, and visualized using the RPT1/biotin-goat anti-mouse IgG/125I-labeled streptavidin system. C, sedimentation profile of AhR from cytosolic extract of Hepa-1 cells treated with 1 nM TCDD for 2 h. Extracts were applied to 10–30% sucrose gradient in MENG and fractionated. Fractions were acetone precipitated and analyzed by SDS-PAGE, visualized as in panel B, and bands excised and quantitated in a gamma counter. D, ratios of AhR to XAP2 in Hepa-1 cells in cytosolic extracts (cytosol) and in the immunoprecipitated 9 S AhR (complex).

Article Snippet: The membrane was then analyzed by Western blot with RPT1 (anti-AhR MAb) and anti-ARA9 (anti-XAP2 MAb; Novus Biologicals) primary antibodies, and 125I-labeled goat anti-mouse IgG (PerkinElmer Life Sciences) secondary Ab.

Techniques: Control, Membrane, Labeling, Sedimentation, SDS Page, Immunoprecipitation

Journal: Advanced Science

Article Title: NARFL Knockout Triggers Ferroptosis‐Driven Vascular Endothelial Dysfunction

doi: 10.1002/advs.202415580

Figure Lengend Snippet: Deletion of the Ciao3 Gene Leads to Embryonic Death and Vascular Development Disorder in Mice. A) The embryo morphology of the Ciao3 heterozygous offspring at 8.5, 10.5, 12.5 and 13.5 days; Ciao3 knockout embryos still existed at 12.5 days but were completely absorbed at 13.5 days and later. B) H&E staining morphology of wild type with 8.5‐day and 12.5‐day mouse embryos and Ciao3 −/− mice embryos with 10.5‐day and 12.5‐day, the development of Ciao3 knock‐out embryos was significantly slower than that of wild‐type mouse embryos and the development of the vascular system was blocked. C) The endothelial marker CD31 was detected by immunofluorescence staining in 12.5‐day mouse embryos. D) The endothelial progenitor cell marker CD34 was detected by immunofluorescence staining in 12.5‐day mouse embryos. E,F) The sections of 12.5‐day‐old mouse embryos were detected by 4‐HNE (E) and BODIPY (F) probes; the positive rate of 4‐HNE and BODIPY in Ciao3 − / − mouse embryo was significantly higher than in wild‐type mouse embryo. H) γ‐H2AX detection of 12.5‐day mouse embryos showed that the DNA damage of Ciao3 − / − mouse embryos was significantly enhanced compared with wild‐type embryos. G‐I) Western blotting showed that GPX4, xCT and FTL were significantly down‐regulated in Ciao3 knockout mouse embryos; the expression of TFR1 and IRP1 was significantly up‐regulated. Data were analyzed by an unpaired Student's t‐test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies for Ciao3 (sc‐514078, 1/1000) were obtained from santa, GPX4 (ab125066, 1/2000) and FTL (ab6990, 1/500) from Abcam, xCT (NB300‐318, 1/2000) and FTH1 (NBP1‐31944, 1/500) from Novus, TFR1 (13‐6800, 1/500) from Invitrogen, GAPDH (ab8245, 1/1000) from Abcam.

Techniques: Knock-Out, Staining, Marker, Immunofluorescence, Western Blot, Expressing

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 1 | Design strategies for endocytosis-triggering EndoTags. a, Schema of designed endocytosis mechanisms. Top, design of binding to constitutively cycling receptors at sites that do not overlap with binding sites for natural ligands to avoid competition. Middle, design of binders that trigger endocytosis by eliciting conformational changes in the receptor. The EndoTag binds at two distinct epitopes on the target and actively triggers the conformational change. Bottom, designed endocytosis via receptor clustering. The multivalent EndoTag clusters multiple copies of the target receptor and induces endocytosis. b, Design strategy for sortilin48 and TfR49 EndoTags. c, Cellular uptake of 100 nM AF647-labelled Sort_EndoTags, TfR-EndoTags or LHDB22 scaffold control for 2 h in U-251MG cells. Data were normalized to the 100 nM AF647-labelled LHDB group (no endocytosis). MFI, mean fluorescence intensity. d, Confocal imaging of Sort_EndoTag (red) and lysosomal marker (green, AF488-labelled LysoTracker) after 24 h incubation in U-251MG cells. e, Design strategy for

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Binding Assay, Control, Fluorescence, Imaging, Marker, Incubation

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 3 | Clearance of soluble proteins by IGF2R pLYTACs. a, Schema for the use of soluble pLYTACs with IGF_EndoTags. b, Cellular uptake of LHDB–AF647 via LHDA–IGF_EndoTags in Jurkat cells. Cells were incubated with 33 nM LHDB– AF647 with or without 1 μM LHDA–IGF_EndoTags for 24 h, washed twice with cold PBS and analysed by flow cytometry. c, Remaining supernatant LHDB– AF647 levels in Jurkat cells. Jurkat cells were incubated with 100 nM LHDB– AF647 with or without 500 nM LHDA–IGF_EndoTags. At timepoints 24 h and 48 h, the cells were pelleted down, and IgG in the supernatant was quantified using a Neo2 plate reader. IgG level was normalized to the IgG-alone control group. d, Cellular uptake of IgG–AF647 via protein G–IGF_EndoTags in K562 cells. Cells were incubated with 33 nM IgG–AF647 with or without 1 μM protein G–IGF_EndoTag3 for 24 h, washed twice with cold PBS and analysed by flow cytometry. The fold change in MFI was calculated by normalizing to the

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Incubation, Flow Cytometry, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Protective effects of Gryllus bimaculatus (Gb) extract on abnormal expression levels of glutamatergic and GABAergic synaptic proteins in the valproic acid (VPA)-induced autism spectrum disorder (ASD) mouse brain tissues. Immunoblot analyses for GRM5, vGluT1, NMDA R1, GABA R1α, and VGAT proteins were performed on prefrontal cortex (PFC) tissue lysates collected at embryonic day 15 (E15) (A) , postnatal day 3 (P3) (B) , and P40 (C) from mice subjected to various treatment combinations. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Expressing, Western Blot, Saline, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Regulatory effects of Gryllus bimaculatus (Gb) extract on excitatory and inhibitory neuronal activity in primary cortical neurons from valproic acid (VPA)-treated embryonic mice. (A) Schematic representation of primary cortical neuron cultures derived from embryonic mouse brains. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (B,D) Immunoblot analyses of NMDA R1, vGluT1, GRM5, GABA R1α, VGAT, NLGN3, NRXN1, and Tuj-1 in cultured primary cortical neuron lysates. Equal amounts of protein were loaded per lane, with β-tubulin used as a loading control. The bars represent fold-changes in the densitometric values of individual protein bands relative to the corresponding β-tubulin band densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant). (C) Confocal microscopy images of cortical neurons from various experimental groups. Cells were cultured for 7 days, fixed, and subsequently immunostained for vGluT1 (red), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm.

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Activity Assay, Derivative Assay, Saline, Western Blot, Cell Culture, Control, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Crucial role of astrocytes in excitatory and inhibitory (E/I) neurotransporter activities in Gryllus bimaculatus (Gb) extract-treated mixed cultures from valproic acid (VPA)-treated mouse brain. (A) Schematic representation of three different types of mixed culture systems derived from embryonic and postnatal mouse brains: Type 1, astrocytes from each treatment group combined with neurons from untreated mice; Type 2, astrocytes from untreated mice combined with neurons from each treatment group; Type 3, astrocytes and neurons both derived from the same treatment group. Astrocytes from postnatal day 3 mouse brains were seeded for 7 days, followed by the addition of cortical neurons from embryonic day 15 mouse brains onto astrocytes monolayers for an additional 7 days. (B–D) Confocal microscopy images of the different types of mixed cultures. Cells were fixed and immunostained for Tuj-1 (green) and GFAP (purple), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (E) Western blots analysis of type III mixed culture. Cell lysates were immunoblotted for Tuj-1, GFAP, synaptophysin, NMDA receptor 1 (NMDA R1), GABA receptor 1α (GABA R1α), EAAT1, and EAAT2. Equal amounts of protein were loaded per each lane, with β-actin serving as the loading control. Bars represent fold-changes in the densitometric values of the bands relative to the corresponding β-actin densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Derivative Assay, Confocal Microscopy, Saline, Western Blot, Control

Journal: Frontiers in Medicine

Article Title: Construction of tissue engineered cornea with skin-derived corneal endothelial-like cell and mechanism research for the cell differentiation

doi: 10.3389/fmed.2024.1448248

Figure Lengend Snippet: SKPs differentiated into CEC-like cells. (A) SKPs were cultured as floating spheres. During cell differentiation, the morphology of the cells changed gradually. On day 10, the cells became the most endothelial-like and formed a mosaic monolayer. (B) Immunofluorescence showed that the CEC-like cells expressed CEC markers Na + /K + ATPase, ZO-1, and Pitx2 after 10 days of differentiation. (C) RT-PCR showed that the CEC-like cells expressed CEC markers Pax6, Cdh2, Car2, Slc4a4, Col4a2 and Col8a2. (Data are mean ± SEM, * p < 0.05, n = 3, scale bar = 100 μm).

Article Snippet: The primary antibodies used included Na + /K + ATPase (1:100, Novus, NB300-146), ZO-1 (1:100, CST, 13663S), PITX2 (1:100, Abcam, ab98297), and human nuclei (1,100, Millipore, MAB1281).

Techniques: Cell Culture, Cell Differentiation, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction