Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Flow cytometry analysis of U937 cells stained with sCD22-ECD alone (red) or sCD22 precomplexed with a decoy peptide comprising the M6P-binding sites (blue) or the IGF2 site (orange) on IGF2R. (B) Time-lapse fluorescence microscopy analysis of cathepsin D trafficking to lysosomes in U937 cells treated with sCD22-Δ (black), sCD22-ECD (red), sCD22-ECD, and anti-IGF2R (blue) or saturating amounts of M6P (brown) (n = 3, ANOVA, means ± SEM). N.S., not significant. (C) Time-lapse fluorescence microscopy analysis of NPC2 trafficking to lysosomes in U937 cells treated with sCD22-Δ (black), sCD22-ECD (red), sCD22-ECD and anti-IGF2R (blue), or saturating amounts of M6P (brown) (n = 2, ANOVA, means ± SEM). (D) Western blot analysis of CTSD proteoform expression in WT and IGF2R KO U937 cells treated with sCD22-ECD or sCD22-Δ for 24 hours. Equal loading was confirmed across lanes by total protein stain (n = 3, one-way ANOVA, means ± SEM). (E) Western blot analysis of NPC2 expression in WT and IGF2R KO U937 cells treated with sCD22-ECD or sCD22-Δ for 24 hours. Equal loading was confirmed across lanes by total protein stain (n = 3, one-way ANOVA, means ± SEM). (F) Representative images of NPC2 (gray) and LAMP2 (green) expression in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (G) Proportion of NPC2+ area to LAMP2+ area in U937 cells treated with sCD22-Δ or full-length sCD22-ECD (n = 8, t test, means ± SD). (H) Representative images of IGF2R (gray) colocalization (Coloc) (yellow) with the Golgi marker GOLGA1 (red) in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (I) Proportion of IGF2R localized to the Golgi in U937 cells treated with sCD22-Δ or sCD22-ECD (n = 3 biological replicates, three to four cells quantified per replicate, t test, means ± SD). (J) Representative images of IGF2R (gray) colocalization (yellow) with the lysosomal marker LAMP1 (cyan) in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (K) Proportion of IGF2R localized to the lysosome in U937 cells treated with sCD22-Δ or sCD22-ECD (n = 3 biological replicates, two to three cells quantified per replicate, t test, means ± SD). (L) Representative images of IGF2R (gray) colocalization (yellow) with wheat germ agglutinin (WGA) cell surface staining (green) in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (M) Proportion of IGF2R localized to the cell surface in U937 cells treated with sCD22-Δ or sCD22-ECD (n = 3 biological replicates, three to four cells quantified per replicate, t test, means ± SD). (N) Flow cytometry analysis of iMGLs treated with sCD22-Δ or sCD22-ECD, incubated with pHrodo-myelin for 24 hours, and stained with BODIPY, with corresponding histograms. (O) Quantification of phagocytosis by pHrodo-myelin mean fluorescence intensity (MFI) in iMGLs treated with sCD22-Δ or sCD22-ECD (n = 4, t test, means ± SEM). (P) Quantification of lipid droplet storage by BODIPY MFI in iMGLs treated with sCD22-Δ or sCD22-ECD (n = 4, t test, means ± SEM).

Article Snippet: Exogenous lysosomal protein recapture assay Recombinant His-tagged CTSD (R&D Systems, 1014-AS) or NPC2 (Sino Biological, 13341-H08H) was conjugated to CypHer5E NHS (GE Life Sciences) as described above.

Techniques: Flow Cytometry, Staining, Binding Assay, Fluorescence, Microscopy, Western Blot, Expressing, Marker, Incubation

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of mAb generation and screening pipeline. (B) Screening results of 38 mAb clones for binding to CD22 (first column) and blocking of sCD22 to IGF2R on cell surface (second and third columns are two independent experiments). Three clones with adequate binding and potent blocking are highlighted (M22, M28, and M42). (C) Association-dissociation curves of antibody candidates binding to CD22 determined by biolayer interferometry. (D) Dose-response curves of CD22-IGF2R blockade by antibody candidates determined by flow cytometry. IC50, median inhibitory concentration. (E) Time-lapse fluorescence microscopy analysis of NPC2 trafficking to lysosomes in U937 cells treated with sCD22-Δ (gray), sCD22-ECD and an isotype control antibody (purple), or sCD22-ECD and clone M42 (green) (n = 2, ANOVA, means ± SEM). (F) Schematic of pipeline to generate isogenic WT and I1061T mutant iMGLs from iPSCs edited by CRISPR-Cas9–directed homologous recombination. After introduction of donor single-stranded DNA (ssDNA) by electroporation, a homozygous T3182C nucleotide substitution was confirmed by Sanger sequencing. NPC1 reduction was confirmed by Western blot. Mutant and isogenic control iPSCs were subsequently directed toward a hematopoietic lineage and differentiated into microglia-like cells. (G) Western blot quantification of NPC1 expression normalized to a loading control (β-actin) in WT and I1061T mutant iPSCs (n = 3, t test, means ± SEM). (H) Representative images of WT and I1061T mutant iMGLs stained for Filipin III (red, unesterified cholesterol) and IBA1 (green, microglia marker). Scale bar, 20 μm. (I) Quantification of Filipin-positive area normalized to total IBA1-positive area in WT (gray) and I1061T mutant (blue) iMGLs (n = 5 biological replicates, t test, means ± SEM). (J) Schematic of human in vitro model of microglia in NPC. Three components (iPSC-derived microglia, I1061T patient mutation, and NPC patient CSF) were combined to test the proof-of-principal in vitro efficacy of anti-CD22 in NPC. (K) Representative images of I1061T mutant iMGLs treated with NPC CSF and an isotype control antibody stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (L) Representative images of I1061T mutant iMGLs treated with NPC CSF and anti-CD22 stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (M) Quantification of Filipin-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (N) Quantification of LAMP2-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (O) Heatmap of normalized counts (z score) for differentially expressed genes in WT and I1061T mutant iMGLs treated with NPC CSF and isotype or anti-CD22. (P) Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. IRE1, inositol-requiring enzyme 1; IFN-γ, interferon-γ; UPR, unfolded protein response. (Q) GO cellular component enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. MHC-II, major histocompatibility complex class II; ER, endoplasmic reticulum.

Article Snippet: Exogenous lysosomal protein recapture assay Recombinant His-tagged CTSD (R&D Systems, 1014-AS) or NPC2 (Sino Biological, 13341-H08H) was conjugated to CypHer5E NHS (GE Life Sciences) as described above.

Techniques: Clone Assay, Binding Assay, Blocking Assay, Flow Cytometry, Concentration Assay, Fluorescence, Microscopy, Mutagenesis, CRISPR, Homologous Recombination, Electroporation, Sequencing, Western Blot, Expressing, Staining, Marker, In Vitro, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Serological cross-reactivity between Crimean-Congo haemorrhagic fever virus and Nairobi sheep disease virus glycoprotein C

doi: 10.3389/fimmu.2024.1423474

Figure Lengend Snippet: Orthonairovirus test antigens employed during experiments.

Article Snippet: NSDV Gc , ACH99800 (708, Kenya) , HEK293 (Human) , The Native Antigen Company, UK #REC31906.

Techniques: Expressing, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: pVHL regulates protein stability of the TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

doi: 10.1007/s00018-025-05852-0

Figure Lengend Snippet: pVHL inhibits Wnt/β-catenin signaling and destabilizes TCF/LEF protein ( A ) TOPFlash luciferase assays in HCT116 cells with increasing pVHL overexpression. Expression of Flag-pVHL was confirmed by western blotting. Values are mean ± S.D. ( n = 3). One-way ANOVA analysis with Dunnett's multiple comparisons test, ** p < 0.01; *** p < 0.001. ( B ) TOPFlash assays in VP16-Tcf7l1ΔN-treated HEK293T cells with increasing pVHL overexpression. Expression of Flag-pVHL was confirmed by western blotting. Wnt/β-catenin signal was activated by transfection with VP16-Tcf7l1ΔN plasmid DNA (50 ng). Expression of Flag-pVHL was confirmed by western blotting. Values are mean ± S.D. ( n = 3). One-way ANOVA analysis with Dunnett's multiple comparisons test, ** p < 0.01; *** p < 0.001; **** p < 0.0001. ( C ) TOPFlash luciferase assays in VHL -knockout HEK293T cells. pVHL protein levels were confirmed by western blotting. Values are mean ± S.D. ( n = 3). Unpaired t -test, * p < 0.05; ** p < 0.01; *** p < 0.001. ( D ) TOPFlash luciferase assays in BIO-treated VHL -knockout HEK293T cells. TOPFlash plasmid was cotransfected with Renilla plasmid into control or VHL -knockout cells. Wnt/β-catenin activity was induced by BIO (1 μM) for 4 h. Values are mean ± S.D. ( n = 3). Unpaired t -test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ( E ) The transcriptional levels of Wnt target gene AXIN2 , NKD1 , and CCND1 in VHL -knockout HEK293T cells were analyzed by qRT-PCR. Values are mean ± S.D. ( n = 3). Unpaired t -test, ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. ( F ) The transcriptional levels of Wnt target gene AXIN2 and NKD1 in pVHL-overexpressing 786-O cells were analyzed by qRT-PCR. Values are mean ± S.D. ( n = 3). Unpaired t -test, ns, not significant; * p < 0.05; ** p < 0.01. ( G ) Endogenous TCF protein levels in HEK293T cells with increasing pVHL overexpression. Western blot analysis detected two distinct TCF7L2 isoforms: TCF7L2E and TCF7L2M/S. (H) Reintroduction of Flag-pVHL downregulated TCF7 and TCF7L2 in 786-O cells. (I) Flag-pVHL promotes endogenous TCF7L2 degradation in HEK293T cells. HEK293T cells were transfected with empty vector or Flag-pVHL, after 48 h, treated with cycloheximide (CHX; 100 μg mL. −1 ), and harvested at indicated time points (0, 2, 4, 8 h). Quantification of the total protein levels of the two TCF7L2 isoforms, TCF7L2E and TCF7L2M/S were normalized to Histone H3 (right panel). Values are mean ± S.D. ( n = 3). Two-way ANOVA analysis with Bonferroni's multiple comparisons test, ns, not significant; * p < 0.05; *** p < 0.001. (J) The protein levels of TCF/LEF in control and VHL -Knockout cells. The expression level of HIF-1α was used as a positive control. Relative protein level normalized to Histone H3 (lower panel). Values are mean ± S.D. ( n = 3). Unpaired t -test, * p < 0.05; ** p < 0.01; *** p < 0.001. (K) The transcriptional levels of TCF/LEF in control and VHL -knockout cells were analyzed by qRT-PCR. Values are mean ± S.D. ( n = 3). Unpaired t -test, ns, not significant. (L) Introduction of Flag-pVHL into VHL -knockout HEK293T cells downregulated TCF/LEF protein levels. Relative protein level normalized to Histone H3 (lower panel). SE, short time of exposure; LE, long time of exposure. Values are mean ± S.D. ( n = 3). Unpaired t -test, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The recombinant proteins for human pVHL (CSB-EP025852HU) and TCF7L2(1–456) (CSB-EP889079HU), expressed in the Escherichia coli expression system, were obtained from CUSABIO (Wuhan, China).

Techniques: Luciferase, Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Knock-Out, Control, Activity Assay, Quantitative RT-PCR, Positive Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: pVHL regulates protein stability of the TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

doi: 10.1007/s00018-025-05852-0

Figure Lengend Snippet: pVHL directly binds with TCF/LEF ( A ) Detection of pVHL binding to TCF/LEF in HEK293T cells by Co-IP. Red asterisk indicates the specific band. ( B ) Co-IP assay revealed the endogenous interaction between TCF7L2 and pVHL in HEK293T cells. ( C , D ) pVHL pulls down TCF/LEF. Purified GST or GST-pVHL proteins were incubated with extracts of HEK293T cells either transfected with Myc-Tcf/Lef ( C ) or untransfected ( D ). Bound proteins were eluted and analyzed by western blot using indicated antibodies. Red asterisk indicates GST-pVHL. ( E ) Surface plasmon resonance analysis of interactions between pVHL with TCF7L2(1–456) using purified recombinant proteins. ( F ) Schematic representations of pVHL and truncated mutant proteins. ( G and H ) Mapping pVHL binding domain interacting with endogenous or exogenous TCF7L2 in transfected HEK293T cells by Co-IP assay. ( I ) Schematic representation of of TCF7L2 WT and truncated mutant proteins. ( J ) Mapping TCF7L2 binding domain interacting with pVHL in transfected HEK293T cells by Co-IP assay

Article Snippet: The recombinant proteins for human pVHL (CSB-EP025852HU) and TCF7L2(1–456) (CSB-EP889079HU), expressed in the Escherichia coli expression system, were obtained from CUSABIO (Wuhan, China).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Purification, Incubation, Transfection, Western Blot, SPR Assay, Recombinant, Mutagenesis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: pVHL regulates protein stability of the TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

doi: 10.1007/s00018-025-05852-0

Figure Lengend Snippet: pVHL promotes TCF/LEF degradation by ubiquitin-independent proteasome pathway ( A ) Changes in endogenous TCF7L2 protein levels in pVHL-overexpressing HEK293T cells treated with indicated inhibitors. The transfected cells were either untreated or treated with MG132 (10 μM), NH 4 Cl (25 mM), or 3-MA (5 mM) for 8 h. ( B ) Effects of pVHL-overexpression on Tcf7l2 ubiquitination. Myc-Tcf7l2, Flag-HIF-1α, and HA-Ub were co-transfected with GFP-Vector or pVHL-GFP into HEK293T cells. After 48 h, cells were treated with MG132 (10 μM) for 8 h, and lysed for immunoprecipitation with anti-Myc and anti-Flag antibody. ( C ) Changes in Tcf7l2-K/R protein levels in pVHL-overexpressing HEK293T cells treated with indicated inhibitors. Western blot analysis of whole cell lysis derived from HEK293T cells transfected with indicated plasmid DNA and either untreated or treated with MG132 (10 μM), NH 4 Cl (25 mM), or 3-MA (5 mM) for 8 h. ( D ) Changes in endogenous TCF7L2 or HIF-2α protein levels in pVHL-overexpressing HEK293T cells treated with a specific inhibitor for the ubiquitin activating enzyme or proteasome. The transfected cells were either untreated or treated with TAK243 (1 μM) for 12 h or MG132 (10 μM) for 8 h. ( E ) Degradation analysis of TCF7L2(1–456) protein in cell-free system. The amount of TCF7L2(1–456) protein degraded by 26S proteasome at each indicated time points (0, 2, 4, 6, 12, 18 h) in cell-free system. The group with MG132 treatment was used as a control. Values are mean ± S.D. ( n = 3). One-way ANOVA analysis with Tukey's multiple comparisons test. Groups marked with distinct letters show significant differences from one another ( p < 0.05)

Article Snippet: The recombinant proteins for human pVHL (CSB-EP025852HU) and TCF7L2(1–456) (CSB-EP889079HU), expressed in the Escherichia coli expression system, were obtained from CUSABIO (Wuhan, China).

Techniques: Ubiquitin Proteomics, Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Western Blot, Lysis, Derivative Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: pVHL regulates protein stability of the TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

doi: 10.1007/s00018-025-05852-0

Figure Lengend Snippet: pVHL promotes TCF degradation in an E3 ubiquitin ligase-independent manner ( A ) Tcf7l2 protein levels in HEK293T cells with overexpression of WT, site-mutated, or truncated pVHL. ( B ) TOPFlash reporter assays in VP16-Tcf7l1ΔN-transfected HEK293T cells with overexpression of WT, site-mutated, or truncated pVHL. Wnt/β-catenin signal was activated by transfection with VP16-Tcf7l1ΔN (50 ng). Values are mean ± S.D. ( n = 3). Unpaired t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) Tcf7l2-K/R protein levels in HEK293T cells with overexpression of WT, site-mutated, or truncated pVHL. ( D ) Tcf7l1-HMG DBD protein levels in HEK293T cells with overexpression of WT, site-mutated, or truncated pVHL. ( E ) pVHL truncation mutant pVHL (1–157) promotes endogenous TCF7L2 degradation in HEK293T cells. HEK293T cells were transfected with empty vector or Flag-pVHL (1–157), after 48 h, treated with cycloheximide (CHX; 100 μg mL −1 ) and harvested at indicated time points (0, 2, 4, 8 h). The total protein levels of the two TCF7L2 isoforms, TCF7L2E and TCF7L2M/S, were normalized to Histone H3 (lower panel). Values are mean ± S.D. ( n = 3). Two-way ANOVA analysis with Bonferroni's multiple comparisons test. ns, not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001. ( F ) Overexpression of Flag-pVHL and Flag-pVHL (1–157) reduced TCF7, TCF7L1, and TCF7L2 protein levels in VHL -KO cells. HIF-1α was downregulated in VHL -KO after transfection with Flag-pVHL but not with Flag-pVHL (1–157). Relative protein level normalized to Histone H3 (right panel). Values are mean ± S.D. ( n = 3). Unpaired t -test, ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. ( G ) Overexpression of pVHL or pVHL (1–157) decreased endogenous Tcf7l2 protein level in wide-type zebrafish embryos at 24 hpf. Protein samples of 4 zebrafish embryos were added in each well. ( H ) vhl -null mutant zebrafish embryos exhibited elevated protein levels of Tcf7l2 at 120 hpf (upper panel). Reintroduction of pVHL or pVHL (1–157) into vhl -null mutant zebrafish embryos reduced Tcf7l2 protein level at 48 hpf (lower panel). Protein samples of 4 zebrafish embryos were added in each well. ( I ) Changes in endogenous TCF7L2 protein levels in pVHL (1–157)-overexpressing HEK293T cells treated with indicated inhibitors. The transfected cells were either untreated or treated with MG132 (10 μM), NH 4 Cl (25 mM), or 3-MA (5 mM) for 8 h. ( J ) The protein levels of TCF/LEF in control or ELOC -Knockdown cells. The expression level of HIF-1α was used as a positive control

Article Snippet: The recombinant proteins for human pVHL (CSB-EP025852HU) and TCF7L2(1–456) (CSB-EP889079HU), expressed in the Escherichia coli expression system, were obtained from CUSABIO (Wuhan, China).

Techniques: Ubiquitin Proteomics, Over Expression, Transfection, Mutagenesis, Plasmid Preparation, Control, Knockdown, Expressing, Positive Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: pVHL regulates protein stability of the TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

doi: 10.1007/s00018-025-05852-0

Figure Lengend Snippet: pVHL directly interacts with 26S proteasome to promote TCF protein degradation ( A ) IP-MS analyzing the proteins interacting with pVHL in HEK293T cells. After transfecting pCS2-Flag or pCS2-Flag-pVHL plasmids into HEK293T cells for 48 h, cells were treated with MG132 (10 μM) for 8 h. Flag-pVHL was then immunoprecipitated using anti-Flag antibody and analyzed by mass spectrometry. Coomassie blue staining of Flag immunoprecipitates revealed proteins interacting with Flag-pVHL. Red arrow indicates Flag-pVHL. Mass spectrometry analysis identified multiple subunits of the 26S proteasome, including PSMD4 (19S regulatory particle) and PSMA4 (20S core particle), which are highlighted in red in the table (lower panel). ( B ) Co-IP assay revealed that Flag-pVHL interacted with 26S proteasome, as indicated by detecting 19S regulatory subunit PSMD4 in HEK293T cells with an antibody against PSMD4. ( C ) pVHL pulls down 26S proteasome, as indicated by detecting 19S regulatory subunit PSMD4 and 20S core particle PSMA4 in HEK293T cells. Purified GST or GST-pVHL protein was incubated with extracts of HEK293T cells. Red arrow indicates GST-pVHL. ( D ) In vitro cell-free GST pulldown assay revealed direct interaction between pVHL and 26S proteasome. Purified GST or GST-pVHL was incubated with recombinant 26S proteasome. Red arrow indicates GST-pVHL. ( E ) Endogenous TCF7L2 protein levels in HEK293T cells with overexpression of indicated pVHL mutants. ( F ) TOPFlash reporter assays in HEK293T cells with coexpression of VP16-Tcf7l1ΔN and each indicated pVHL mutant. Wnt/β-catenin signal was activated by transfection with VP16-Tcf7l1ΔN (50 ng). Values are mean ± S.D. ( n = 3). One-way ANOVA analysis with Dunnett's multiple comparisons test. ns, not significant; * p < 0.05; **** p < 0.0001. ( G ) In vitro cell-free GST pulldown assay detected binding of full-length and mutant pVHL to 26S proteasome. Red arrows indicate GST-fusion protein. ( H ) Endogenous TCF7L2 protein levels in HEK293T cells with overexpression of indicated pVHL mutants. ( I ) TOPFlash reporter assays in HEK293T cells with coexpression of VP16-Tcf7l1ΔN and each indicated pVHL mutant. Wnt/β-catenin signal was activated by transfection with VP16-Tcf7l1ΔN (50 ng). Values are mean ± S.D. ( n = 3). One-way ANOVA analysis with Dunnett's multiple comparisons test. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ( J ) Introduction of pVHL and pVHL(54–157) into VHL -KO cells reduced TCF7, TCF7L1, and TCF7L2 protein levels. HIF-1α was downregulated in VHL -KO after transfection with pVHL-GFP but not with pVHL(54–157)-GFP. Relative protein level normalized to Histone H3 (lower panel). Values are mean ± S.D. ( n = 3). Unpaired t -test, ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ( K ) Schematic illustration of the mechanism by which TCF/LEF protein stability is regulated by pVHL

Article Snippet: The recombinant proteins for human pVHL (CSB-EP025852HU) and TCF7L2(1–456) (CSB-EP889079HU), expressed in the Escherichia coli expression system, were obtained from CUSABIO (Wuhan, China).

Techniques: Protein-Protein interactions, Immunoprecipitation, Mass Spectrometry, Staining, Co-Immunoprecipitation Assay, Purification, Incubation, In Vitro, GST Pulldown Assay, Recombinant, Over Expression, Mutagenesis, Transfection, Binding Assay

Journal: Science Advances

Article Title: DKK3-LRP1 complex and a chemical inhibitor regulate Aβ clearance in models of Alzheimer’s disease

doi: 10.1126/sciadv.adz2099

Figure Lengend Snippet: ( A and B ) Subcellular colocalization of fluorescently labeled Aβ 42 (FAM-Aβ 42 ; green) with LysoTracker (red) in SH-SY5Y neuroblastoma cells and C8-D1A astrocytic cells following treatment with CM as indicated. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 50 μm. ( C ) Schematic of the experimental protocol used for culturing and differentiating neural stem cells derived from a patient with AD (AD-NSCs). ( D ) Images showing internalization of FAM-Aβ 42 (green) in differentiated AD-NSCs, colabeled with Tuj1 for neurons (red; top) or glial fibrillary acidic protein (GFAP) for astrocytes (red; bottom) under various CM treatments. Scale bar, 50 μm. Tuj1, β-III tubulin. ( E ) ELISA quantification of Aβ 42 uptake in cells treated with CTL, DKK1, or DKK3 CM containing soluble Aβ 42 . ( F ) Similar setup as in (E) but with Aβ 42 preincubated for 2 hours to allow for internalization, followed by a wash and an additional 8-hour incubation in fresh medium. ELISA quantified the decrease in internalized Aβ 42 as an estimate of cellular Aβ clearance (see Materials and Methods for details). Data are presented as mean ± SD. Statistical significance determined by Student’s t test, with ** P < 0.01, *** P < 0.001, and n.s. (not significant).

Article Snippet: Human iPSC-derived NSCs (Axol Bioscience, ax0111) were maintained according to the manufacturer’s protocols on Laminin-coated chamber slides (Thermo Fisher Scientific, 154526).

Techniques: Labeling, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Science Advances

Article Title: DKK3-LRP1 complex and a chemical inhibitor regulate Aβ clearance in models of Alzheimer’s disease

doi: 10.1126/sciadv.adz2099

Figure Lengend Snippet: ( A ) Differentiated AD-NSCs were incubated with 1 μM Aβ 42 and various concentrations of purified DKK1-Flag or DKK3-Flag for 30 min, followed by IP of LRP1 and immunoblotting using specific antibodies. Five percent of the total lysate was used as input, with IgG serving as a negative CTL. ( B ) SH-SY5Y cells stably expressing mLRPIV treated as in (A) but with an expanded range of DKK1 or DKK3 concentrations. Levels of Aβ 42 following HA-tag IP were quantified using ELISA. ( C ) SPR analysis depicting the competitive binding dynamics of Aβ 42 to mLRPIV in the presence of 35.5 nM DKK3. The assay was conducted with varying concentrations of Aβ 42 . ( D ) IF imaging demonstrating the localization of DKK3-Flag (red) and mLRPIV-HA (green) in SH-SY5Y cells at 0, 30, and 60 min posttreatment with DKK3-Flag CM. Arrow indicates cell membrane localization; arrowhead points to intracellular localization. Nuclei are counterstained with DAPI (blue). Scale bar, 50 μm. ( E ) Immunoblot analysis showing levels of Mem and Cyt mLRPIV in SH-SY5Y cells after treatment with 50 nM recombinant DKK1 or DKK3 at specified time points. Na + - and K + -dependent ATPase (Na + ,K + -ATPase) and ACTIN were used as loading controls for membrane and cytoplasmic proteins, respectively. ( F ) Quantification of band density for proteins shown in (E). Membrane and cytoplasmic proteins were normalized to Na + ,K + -ATPase or ACTIN, respectively. Error bars represent mean ± SD from biological triplicates.

Article Snippet: Human iPSC-derived NSCs (Axol Bioscience, ax0111) were maintained according to the manufacturer’s protocols on Laminin-coated chamber slides (Thermo Fisher Scientific, 154526).

Techniques: Incubation, Purification, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Imaging, Membrane, Recombinant