anti tgn46 polyclonal antibody  (Bio-Rad)

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Phylogenetic tree of mammarenavirus GPC amino acid sequences. Tree generated via MUSCLE alignment, Gblock curation, PhyML phylogeny, and rendered with TreeDyn using phylogeny.fr. (B) Enrichment of guide RNAs from a CRISPR-Cas9 loss-of-function genome-wide screen after the second round of infection with chimeric VSV-eGFP-PICV (CoAN3739). Genes arranged by rank on the x-axis; significance (positive score) on the y-axis. (n=1, experimental replicate). (C) WT A549, CD164 KO , and CD164 KO +CD164 cells were infected with WT or chimeric VSV-eGFP expressing indicated glycoproteins (MACV, LCMV, PICV CoAN4763, PICV CoAN3739, PARV, FLEXV) at MOI=3. Six hours post-infection, % GFP-positive cells determined by flow cytometry (n=3, independent experiments in triplicate and technical replicates in duplicate). One-Way ANOVA: * p <0.05, ** p <0.005, *** p <0.0005. (D) WT A549, CD164 KO , and CD164 KO +CD164 cells were used for plaque assays with WT LCMV (Armstrong), PICV (CoAN4763), and PARV. Dotted line indicates limit of detection (LOD). (n=3, independent experiments in triplicate). One-way ANOVA with Tukey’s multiple comparisons: *p< 0.05, **p< 0.005, ***p< 0.0005.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Generated, CRISPR, Genome Wide, Infection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Left: domain architecture of CD164 and deletion mutants; predicted N-linked glycosylation sites (purple), O-linked sites (grey). Right: schematic comparing CD164 and CRD-LAMP1 chimeric protein (CD164 CRD: blue; LAMP1 proximal domain: red). (B) CD164 KO HeLa cells expressing empty vector, CD164, CD164 deletion mutants (ΔMD1, ΔCRD, ΔMD2), or CRD-LAMP1 were inoculated with chimeric VSV-eGFP expressing indicated glycoproteins (MOI=2). % GFP-positive cells determined 6 hpi by flow cytometry; normalized to WT HeLa cells (Relative Infection) (n=3, independent experiments in triplicate and technical replicates in duplicate). One-Way ANOVA: * p <0.05.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Glycoproteomics, Expressing, Plasmid Preparation, Flow Cytometry, Infection

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Acid by-pass assay in CD164 KO HeLa cells expressing plasma membrane-targeted CD164 (WT-CD164 PM ). VSV-eGFP-PICV3739 was bound to Bafilomycin A1-pretreated cells (MOI=3, 4°C, 1 hr) then fusion was triggered at indicated pH, media replaced, and infection proceeded for 5 hr in Bafilomycin A1. % GFP-positive cells normalized to WT HeLa at pH 5.0 (n=3, independent experiments in triplicate and technical replicates in duplicate). (B) Same assay as in (A) performed with WT, CD164 KO cells expressing empty vector, or WT-CD164 PM cells, with fusion triggered at pH 5.2. % GFP-positive cells normalized to WT HeLa (dotted grey line) (n=3, independent experiments in triplicate and technical replicates in duplicate). One-Way ANOVA: * p <0.05, ** p <0.005, *** p <0.0005. (C) WT or CD164 KO A549 cells pretreated with cycloheximide were inoculated with VSV-P/eGFP-PICV3739 (MOI=400, 3 hr). Cells were fixed, stained with DAPI, WGA, anti-VSV-M, and imaged by Airyscan confocal microscopy. Representative cell shown per condition. VSV-M: red; P/eGFP: green; colocalization: yellow; WGA: white; DAPI; blue. Scale bar, 10 µm. (D) Quantification of P/eGFP and VSV-M colocalization from (C) (20 cells per condition). One-Way ANOVA (left) or Kruskal-Wallis ANOVA (right), * p <0.05, ** p <0.005, *** p <0.0005..

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Expressing, Clinical Proteomics, Membrane, Infection, Plasmid Preparation, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: Additional representative images from . WT or CD164 KO A549 cells pretreated with cycloheximide or DMSO, inoculated with VSV-P/eGFP-PICV3739 (A, 3 hr) or VSV-P/eGFP-G (B, 3 h). Cells were fixed, stained with DAPI (blue), WGA (Alexa 647, white), anti-VSV-M (Alexa 594, red), imaged by Airyscan confocal microscopy. Scale bar, 10 µm. (C) Widefield microscopy of WT or CD164 KO A549 pretreated with cycloheximide, inoculated with VSV-P/eGFP-PICV3739, fixed, and stained at 2 hours post-infection.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Staining, Confocal Microscopy, Microscopy, Infection

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Binding of 2.5 µM PICV sGP1 to 100 nM immobilized sCD164 as a function of pH; baseline corrected to binding of MACV sGP1 at same pH (n=3, independent experiments in triplicate). (B) Final binding response of 2.5 µM PICV, PARV, FLEXV, and MACV sGP1 to sCD164 at pH 5.0 (blue) or 7.4 (grey) (n=3, independent experiments in triplicate). One-Way ANOVA: * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.00005. (C) pH 5.0 binding curve of PICV sGP1 to sCD164 (100 nM). Left: 1:1 Langmuir fit; middle: nonlinear fit; right: Scatchard plot. Binding affinity (K D ) calculated from 1:1 Langmuir fit, K D = 0.259 µM ± 0.001 µM. (n=3, independent experiments in triplicate). (D) pH 5.0 binding of PICV sGP1 to soluble sCRD (200 nM). Left: 1:2 Langmuir fit; middle: nonlinear fit; right: Scatchard plot. Binding affinities (K D ) calculated from 1:2 Langmuir fit K D1 = 1.144 μM ± 0.019 µM, and K D2 = 31.86 μM ± 0.555 µM. (n=3, independent experiments in triplicate) (E) Mean fluorescence intensity (MFI/10,000) of AF647-PICV sGP1 binding to CD164 KO +WT-CD164 PM or CD164 KO cells at pH 6.0 or 7.4 (n=3, independent experiments in triplicate). One-Way ANOVA of AUC: *p< 0.05, **p< 0.005, ***p< 0.0005, ****p< 0.00005.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Coomassie-stained SDS-PAGE of soluble proteins used in . 2.5 µg of PNGaseF treated, nondenatured, or denatured purified protein. (B) Nonlinear fit of binding curves at pH 5.0 (blue) and 7.4 (grey) for PICV sGP1, PARV sGP1, FLEXV sGP1, and MACV sGP1 to 100 nM of immobilized sCD164. Curves represent the averaged final response at the end of association (n=3, independent experiments in triplicate). (C) Binding of 5 µM MACV sGP1 (maroon triangle) or 1 µg mouse anti-human TfR1 (CD71) antibody (cyan diamond) to 100 nM of immobilized hTfR1-Fc as a function of pH. Normalized to max binding at pH 8.0 (n=2, independent experiments in duplicate). (D) pH 7.4 binding curve of MACV sGP1 to hTfR1 (100 nM). Left: 1:1 Langmuir fit; middle: nonlinear fit; right: Scatchard plot. Binding affinity (K D ) calculated from 1:1 Langmuir fit, K D = 2.52 µM ± 0.006 µM. (n=2, independent experiments in duplicate). (E) Representative histogram of mean fluorescence intensity (MFI) from . Cells bound with 2.5 µM AF647-labeled PICV sGP1 at pH=6.0 (orange: CD164 KO +WT-CD164 PM ; purple: CD164 KO ) or pH 7.4 (red: CD164 KO +CD164 PM ; blue: CD164 KO ) (n=3, independent experiments in triplicate) (F) MFI (/10,000) of CD164 KO +WT-CD164 PM or CD164 KO cells from and S4E, labeled with 5 µM AF647-PICV sGP1 at pH 6.0 and then washed at pH 7.4 (hashed) or 6.0 (solid). (n=3, independent experiments in triplicate).

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Staining, SDS Page, Purification, Binding Assay, Fluorescence, Labeling

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Alphafold3 predicted structure of PICV sGP1 (yellow) interacting with the CRD of CD164 (blue). Left: full predicted structure; right: zoom on predicted interaction region. Interface Predicted Template Modeling (ipTM) score=0.81. (B) DMS of CRD identifies key determinants for the acid-dependent GP1 interaction. DMS selection for loss of GP1 binding at pH 6.0 (GP1-sorted population S5A). Top: heat map of mutation differential scores (dms_tools2); blue: enriched mutations (>0), red: de-enriched (<0). Bottom: total enriched positive changes per amino acid site (score>0). (n=1, experimental replicate). (C) Averaged final association response of PICV sGP1 binding to sCD164 containing indicated mutations in CRD. Blue: response >WT sCD164 (0.066, white); red: response WT (white, 1.0); red:

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Selection, Binding Assay, Mutagenesis, Expressing, Infection

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Reducing/denaturing or non-denaturing Coomassie of sCD164 mutant proteins . Asterisk (*) denotes denaturing. (B) Final binding response of 1 µg N6B6 anti-human CD164 antibody to 100nM immobilized sCD164 mutants. (C) Inhibition of chimeric VSV expressing indicated glycoproteins by anti-CD164 monoclonal antibody, N6B6. Cells were pretreated with N6B6 for 1 hour on ice, virus was then added at MOI=1. At 6 hours post-infection, cells were fixed and % GFP-positive cells was determined by flow cytometry. % GPF+ was normalized to HeLa cells infected with no inhibition (n=2). (D) Relative expression of CD164 in CD164 mutant addback cells. Top: area under the curve (CD164/actin) compared to WT; bottom: representative Western blot. (E) Representative histograms of total CD164 expression in CD164 mutant addback cells.

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Mutagenesis, Binding Assay, Inhibition, Expressing, Virus, Infection, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: CD164 is an endolysomal host factor for entry of Clade A New World Arenaviruses

doi: 10.64898/2026.04.21.719929

Figure Lengend Snippet: (A) Impact of alanine mutations within the predicted ß-sheet of PICV sGP1 on binding to CD164. Binding of 2.5 µM WT or mutant PICV sGP1 to 100 nM of immobilized sCD164. Averaged response over association and dissociation measured by BLI. (n=3, independent experiments in triplicate). (B) Plaque assays in WT and CD164 KO A549 infected with VSV expressing WT PICV GPC (yellow), K213A mutant (orange), F215A-N216A mutant (pink). Dotted line: LOD. (n=3, independent experiments in triplicate). Two-tailed unpaired t-test, of CD164 KO compared to WT infected with indicated virus: *p< 0.05, **p< 0.005, ***p< 0.0005. (C) Impact of stabilizing ß-sheet mutations (predicted by Protein MPNN) on CD164 binding, measured by BLI as in (A) (n=3, independent experiments in triplicate). (D) Impact of new consensus sequence generated by Protein MPNN analysis (RCTRSC, orange) on GPC-mediated infection by plaque assay in WT and CD164 KO A549 cells as in (B) (n=3, independent experiments in triplicate). Two-tailed unpaired t-test, of CD164 KO compared to WT infected with indicated virus: *p< 0.05, **p< 0.005, ***p< 0.0005

Article Snippet: Alternatively, cells were stained with 1:1000 dilution of a polyclonal sheep anti-human CD164 antibody (“polyclonal antisera,” R&D systems, AF5790) at pH 7.4.

Techniques: Binding Assay, Mutagenesis, Infection, Expressing, Two Tailed Test, Virus, Sequencing, Generated, Plaque Assay

Journal: PLOS One

Article Title: Minigenes for heterologous expression of human and mouse cationic trypsinogen

doi: 10.1371/journal.pone.0343840

Figure Lengend Snippet: A , Secreted trypsinogen protein (arrow) in the conditioned medium 24 hours and 48 hours after transfection was assessed by SDS-PAGE and Coomassie Blue staining. Representative gels from 3 independent transfections are shown. B , Trypsinogen levels in the conditioned medium were determined by trypsin activity measurement after activation with enteropeptidase. Individual values from 4 transfections with duplicates (n = 8) are shown, with the mean and SD indicated. C , Western blot analysis of trypsinogen (arrow) levels in cell lysates 48 hours post-transfection. Alpha-tubulin was measured as loading control. Representative blots are shown. D , PRSS1 mRNA levels were measured 48 hours after transfection by reverse-transcription quantitative PCR and expressed as fold change relative to the average value of the cDNA construct. Individual values from 3 transfections with duplicates (n = 6) are shown, with the mean and SD indicated. E , Splicing of PRSS1 mRNA expressed from cDNA and minigene constructs was analyzed 48 hours after transfection by reverse-transcription PCR and agarose gel electrophoresis. The arrow indicates the correctly spliced PRSS1 band. The smaller faint band in the minigene samples indicated by the asterisk corresponds to an aberrant splice product in which nucleotide c.40 was spliced to c.114 resulting in the deletion of the mini-intron and an extra 74 nucleotides from exon 2.

Article Snippet: The anti-PRSS1 sheep polyclonal antibody (catalog number AF3848, R&D Systems) was used at 1:5000 dilution.

Techniques: Transfection, SDS Page, Staining, Activity Assay, Activation Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Construct, Agarose Gel Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: Investigation of the E1 and B3 nanobodies' interaction with native CD109 using biolayer interferometry. A and B , the interaction between Nb E1 or B3 and CD109 was investigated using biolayer interferometry (BLI). Nb E1 or B3 were immobilized onto biosensors using amine-reactive chemistry and incubated with native soluble CD109 at concentrations from 12.5 to 200 nM. The experimental curves ( solid lines ) are shown with a baseline reference subtracted (from an Nb-coated biosensor kept in buffer without CD109). Nonspecific binding of CD109 to the blank AR2G biosensors is shown in A and was at most 0.05 nm, showing that most of the response in this experiment was specific. Fitted curves ( dotted lines ) were obtained by the approach given in and all fitted constants are given in and .

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: Bivalent binding of immobilized CD109 by E1-Fc and B3-Fc enhances their functional affinity. A and B , the interaction between immobilized E1-Fc or B3-Fc and soluble CD109 was investigated using biolayer interferometry (BLI). E1-Fc ( A ) and B3-Fc ( B ) were immobilized onto anti-human Fc capture (AHC) biosensors and incubated with native CD109 at concentrations from 12.5 to 200 nM for E1-Fc or 12.5 to 800 nM for B3-Fc. The experimental curves ( solid lines) are shown with a baseline reference subtracted (from an Nb-Fc–loaded biosensor kept in buffer without CD109). Nonspecific binding of CD109 to the blank AHC biosensors is shown in B and was at most 0.15 nm, showing that most of the response in this experiment was specific. C and D , the interaction between native CD109 immobilized on a biosensor to soluble E1-Fc and B3-Fc was investigated using BLI. CD109 (10 μg/ml) was immobilized onto AR2G biosensors and subsequently incubated with E1-Fc ( C ) or B3-Fc ( D ) at concentration from 25 to 100 nM. Nonspecific binding of Nb-Fc's to blank AR2G biosensors is shown in C and was at most 0.05 nm. A – D , fitted curves (dotted lines) were obtained by the approach given in and all fitted constants are given in , , , and .

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Binding Assay, Functional Assay, Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: The E1 and B3 nanobodies co-elute with CD109 during size-exclusion chromatography. A , the binding of Nb E1 to CD109 in its native conformation and Nb B3 to both native CD109 and cleaved CD109 (CD109-TEV) was analyzed using SEC. CD109 and nanobodies were incubated together for 30 min at room temperature before loading onto the SEC column. CD109:Nb E1 eluted earlier than CD109 alone, whereas CD109:Nb B3 and CD109-TEV:Nb B3 eluted at the same volume as CD109 and CD109-TEV alone, respectively. B , SDS-PAGE analysis of the fraction containing most protein from each SEC peak confirmed that Nb E1 co-eluted with CD109, while Nb B3 co-eluted with both CD109 and CD109-TEV. C , the binding of E1-Fc and B3-Fc to CD109 was analyzed using SEC. CD109 and Nb-Fc's were incubated for 30 min at room temperature before SEC analysis. CD109 pre-incubated with Nb-Fc's eluted earlier than CD109 alone. The CD109:E1-Fc complex eluted as two peaks, suggesting 1:2 (Nb-Fc:CD109) and 1:1 complex formation, respectively. In contrast, the CD109:B3-Fc complex primarily eluted as a single peak, indicating predominant 1:1 complex formation. In conclusion, E1 and B3, in both their Nb and Nb-Fc fusion forms, bind CD109 and remain associated during SEC.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Size-exclusion Chromatography, Binding Assay, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: E1 and B3 antagonize CD109's interaction with proteases. A and B , reducing SDS-PAGE showing the effect of E1 ( A ) B3 ( B ) and a negative control nanobody ( C ) on the proteolytic cleavage of CD109. CD109 was pre-incubated with a 1:0.5 to 1:3 CD109:Nb molar ratio for 30 min at room temperature and digested with Cy5-labeled chymotrypsin (CT) for 15 min at 37 °C. Chymotrypsin was inhibited with 2 mM PMSF for 15 min at room temperature before denaturation and reduction. The observed MW variations of the Cy5-chymotrypsin-CD109 conjugation bands reflect the autolytic digestion of chymotrypsin into peptides of different sizes, which are separated upon denaturation. The Cy5-fluorescent image of the gel, shown below the Coomassie-stained gel, shows that when CD109 is incubated with chymotrypsin without nanobodies present, Cy5-labeled chymotrypsin is detected in high molecular weight (MW) bands corresponding to CD109-chymotrypsin conjugation products. A , the addition of E1 results in a decrease in the abundance of conjugation products. B , B3 prevents both CD109 cleavage (as determined by Coomassie staining) and CD109's conjugation of chymotrypsin (as determined by Cy5-fluorescence). C , the negative control nanobody does not affect cleavage or protease conjugation. D and E , to determine whether E1 increased the extent of CD109 cleavage by chymotrypsin, CD109 was pre-incubated with a 1:2 CD109:Nb molar ratio of E1 ( D ) or negative control nanobody ( E ), then cleaved with a serial titration of chymotrypsin (from 0.0125 to 0.4 M ratios of chymotrypsin to CD109) for 60 min at 37 °C, and finally inhibited with 2 mM PMSF before analysis by reducing SDS-PAGE. The Cy5-fluorescence images show that E1 decreased conjugation of chymotrypsin by CD109, and the Coomassie-stained images show that CD109 is approximately 8-fold more readily cleaved when bound by E1, consistent with prevention of its chymotrypsin inhibition by E1.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: SDS Page, Negative Control, Incubation, Labeling, Conjugation Assay, Staining, High Molecular Weight, Fluorescence, Titration, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: E1 and B3 epitopes identified by negative stain electron microscopy. A , 3D reconstructions of CD109 ( center ), CD109:E1 ( middle ), and ( right ) CD109:B3 obtained via negative stain electron microscopy (nsTEM). Examples of the exposure images and selected 2D classes from which these are derived are given in A . B , the native CD109 structure (PDB ID 8S3O , colored as in A ) was fitted into the nsTEM-derived 3D reconstructions. Additional densities were observed for CD109:E1 and CD109:B3. Predicted models were generated using AlphaFold3 for the E1 nanobody interacting with the TE domain and the B3 nanobody interacting with the MG4 domain as shown in . These models are grafted onto native CD109 (with the nanobody colored red ), showing a single representative model for E1 and five models for B3 which showed more variation in its AlphaFold3-derived output models. C and D , AlphaFold3-derived models showing E1 ( red , CDRs in yellow ) interaction with the TE domain ( blue ) and B3 ( red , CDRs in yellow ) interacting with the MG4 domain ( green ).

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Staining, Electron Microscopy, Derivative Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: CD109 structure and function. A , the domain organization of CD109. The bait region (BR) sequence with identified cleavage sites ( , , ), the thiol ester ( yellow circle , TE), the furin cleavage site, and the GPI anchor are highlighted. B , a schematic illustration of the protease-inhibitory mechanism of CD109. A protease cleaves the bait region of CD109, triggering a conformational change. During the conformational change, a previously hidden thiol ester is exposed, allowing it to conjugate the protease. This structural rearrangement disrupts noncovalent interactions between the MG8 domain and the rest of CD109, leading to the release of CD109 from the cell surface. C and D , cartoon representation of native and activated CD109 structures determined by cryo-EM (PDB accession codes 8S3O and 9FX3). Domain colors correspond to those in ( A ). The position of the thiol ester is indicated in both structures. The bait region is not modeled in native CD109 but the 55 Å distance that it spans between Asp649 and His689, indicated with a dotted red line . The cavity that is occupied by trapped proteases is indicated by an orange circle in cleaved CD109. Note that in these structures, CD109 is oriented with its TE domain and MG8 domain facing upwards, which is upside-down compared to its orientation in ( B ).

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Sequencing, Cryo-EM Sample Prep

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: Schematic illustrations of the E1 and B3 mechanisms of action. A , the protease-inhibitory mechanism of CD109. A protease cleaves the bait region of CD109, triggering a conformational change. During the conformational change, a previously hidden thiol ester is exposed, allowing it to conjugate the protease. This structural rearrangement disrupts noncovalent interactions between the MG8 domain and the rest of CD109, leading to the release of CD109 from the cell surface. B , E1's mechanism of action. E1 binds to the TE domain. When a protease cleaves the bait region, E1 temporarily stabilizes the native conformation, slowing down the conformational change. This delay allows the protease to diffuse away, favoring hydrolysis of the thiol ester over conjugation of proteases. C , B3's mechanism of action. Nb B3 binds in the vicinity of the bait region and sterically hinders proteases from accessing the bait region and cleaving CD109.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Conjugation Assay

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: E1-Fc and B3-Fc antagonize CD109's inhibition of chymotrypsin. A , CD109 was incubated with E1-Fc or B3-Fc in a (1:1) or (1:2), respectively, molar ratio of CD109:Nb-Fc, or buffer only for 30 min at room temperature. Chymotrypsin (#C3142, Sigma-aldrich) was added to the indicated ratios of CD109 in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl2, pH 8 for 15 min, after which DQ-labeled gelatin was added as a chymotrypsin substrate. The fluorescence of digested gelatin was measured after 20 min. Chymotrypsin activity is normalized to its activity without the addition of CD109 or Nb-Fc. B , chymotrypsin activity at a 0:1 and 32:1 CD109:chymotrypsin molar ratio, with or without the addition of Nb-Fc. The significance of the inhibition of chymotrypsin by CD109 and the antagonism of CD109's inhibition by E1-Fc and B3-Fc was tested by unpaired two-tailed t-tests, and the resulting p -values are shown on the diagram. Chymotrypsin was significantly inhibited by CD109, and E1-Fc and B3-Fc significantly antagonized this inhibition. Both Fc constructs increased the activity of chymotrypsin when added without CD109 (approximately 20% increase), likely due to stabilizing chymotrypsin before gelatin addition. This stabilization is also conveyed by CD109 and can therefore be neglected when comparing the effect of CD109 with CD109-antibody complexes. Data are shown as the mean values ± SD, n = 3 (technical replicates). C , the ability of E1-Fc and B3-Fc to prevent CD109's chymotrypsin inhibition was investigated using α2-macroglobulin (A2M) as the protease substrate. CD109 was incubated with B3-Fc at a 1:2 M ratio of CD109: B3-Fc or with E1-Fc at a 1:1 M ratio for 15 min. Chymotrypsin was then added to a 1:18 protease:CD109 M ratio for 10 min at 37 °C. Then, A2M was added (using an amount of A2M giving a 1.2:1 chymotrypsin:A2M M ratio) and digested for an additional 5 min at 37 °C. Chymotrypsin was then inhibited with 2 mM PMSF prior to PAGE analysis. Controls were included which added buffer instead of CD109, antibody, and/or chymotrypsin but otherwise kept conditions identical. The samples were then analyzed by pore-limited native PAGE to determine the conformation of A2M. CD109 inhibited chymotrypsin's cleavage of A2M, and E1-Fc and B3-Fc antagonized this inhibition allowing chymotrypsin to cleave A2M despite pre-incubation with CD109.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Inhibition, Incubation, Labeling, Fluorescence, Activity Assay, Two Tailed Test, Construct, Clear Native PAGE

Journal: The Journal of Biological Chemistry

Article Title: Antagonist nanobodies prevent protease inhibition by CD109

doi: 10.1016/j.jbc.2026.111187

Figure Lengend Snippet: B3-Fc blocked the release of CD109 from the cell surface. A , HEK293F cells were transfected with membrane-bound CD109. Three hours post-transfection, cells were treated with 50 nM Nb-Fc's or media alone. This treatment was administered four times in total, and cells were harvested on day 4. B , cell media was then analyzed by Western blotting with a polyclonal anti-CD109 antibody (R&D Systems, #AF4385). The Western blot results show that when cells were treated with B3-Fc, N- and C-terminal fragments were not present in the media, whereas they were detected in cells treated with E1-Fc or media alone. This suggests that B3-Fc prevents the protease-mediated release of CD109 from the cell surface. Note that the human Fc regions of the Nb-Fc fusions were nonspecifically bound by the anti-sheep antibody-HRP conjugate used for detection. C , a schematic illustration of B3 and E1 mechanisms of action on cells. B3-Fc binds near the bait region of membrane-bound CD109 and blocks protease cleavage, preventing the protease-mediated release of CD109 from the cell surface. E1-Fc binds to the TE domain on CD109 and when an incoming protease cleaves the bait region, E1-Fc prevents conjugation of the protease and thus the protease remains active. E1-Fc dissociates from CD109 after CD109 is cleaved.

Article Snippet: The blots were blocked in 5% milk for 2 h at room temperature and incubated with primary antibody, polyclonal sheep anti-CD109 antibody (R&D Systems, product #AF4385), overnight at 4 °C.

Techniques: Transfection, Membrane, Western Blot, Conjugation Assay