mouse anti hcfc1  (Novus Biologicals)

Journal: bioRxiv

Article Title: HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

doi: 10.64898/2026.03.19.712392

Figure Lengend Snippet: ( A ) Schematic model of HCF1-OGT complex binding to D/EHxY peptides. An AlphaFold3 model of KANSL3 DHSY peptide bound to HCF1 is shown. ( B ) Design of proteome-wide HBM library by assembly of D/EHxY-containing peptides from the intrinsically disordered regions (IDRs) of human proteins. ( C ) Scheme of bacterial surface peptide display experiment used to measure binding of Kelch domain of HCF1 EGFP fusion to E. coli cells displaying HCF1 binding peptides fused to MYC-tagged eCPX protein. ( D ) Flow cytometry plots showing HCF1(Kelch)-EGFP and Alexa-647 anti-MYC staining of peptide displaying E. coli cells. A representative experiment of two biological replicates is shown. ( E ) Corrected log2 fold change and p-values of D/EHxY peptides for HCF1(Kelch)-EGFP binding in pooled peptide display experiments compared to their alanine control peptides. ( F ) Western blot of a peptide pulldown experiment testing the indicated peptide GFP fusions in pulling down HCF1 from HCT116 cell lysate. A representative example of two biological replicates is shown. ( G ) Distribution of enrichment scores for HCF1(Kelch)-EGFP binding in a pooled peptide display experiment using libraries of 347 D/EHxY peptides or 259 N/QHxY peptides together with their D/E/N/QHxA negative control mutants. ( H ) Sequence logos showing the positional preferences in different groups of D/EHxY peptides.

Article Snippet: To test the interaction between recombinant HBM peptides and HCF1, the peptide-GFP proteins were loaded onto ChromoTek GFP-Trap® Magnetic Agarose (Proteintech), followed by incubation with human cell lysate containing endogenous HCF1.

Techniques: Binding Assay, Flow Cytometry, Staining, Control, Western Blot, Negative Control, Sequencing

Journal: bioRxiv

Article Title: HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

doi: 10.64898/2026.03.19.712392

Figure Lengend Snippet: ( A ) AlphaFold3 model of HCF1 Kelch domain (blue) and THAP11 DHxY peptide complex. The HCF1 153-169 β-sheets are not shown in order to visualize the peptide. ( B ) Alignment of HCF1 binding peptides with the relative solvent accessible areas (peptide in complex with HCF1 compared to unbound peptide) shown for each residue. ( C ) Wild-type-corrected HCF1 binding scores measured in bacterial peptide display with deep mutational scanning libraries of KANSL3, DIDO1, SETD1A, and UL48 peptides. Below, sequence logos showing favored and disfavored residues for HCF1 binding in each position of the peptides. ( D ) The Gini index (selectivity) showing inequality between residues in a position, calculated from DMS data, and the number of van der Waals contacts of the residue with HCF1 in AlphaFold3 model. ( E ) Dose-response curves showing the ability of different DIDO1 peptide variants as competitor peptides to displace a FITC-labeled HCF1 binding peptide from HCF1, measured by fluorescence polarization (n = 2 biological replicates).

Article Snippet: To test the interaction between recombinant HBM peptides and HCF1, the peptide-GFP proteins were loaded onto ChromoTek GFP-Trap® Magnetic Agarose (Proteintech), followed by incubation with human cell lysate containing endogenous HCF1.

Techniques: Binding Assay, Solvent, Residue, Sequencing, Labeling, Fluorescence

Journal: bioRxiv

Article Title: HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

doi: 10.64898/2026.03.19.712392

Figure Lengend Snippet: ( A ) Scoring of all D/EHxY peptides in the intrinsically disordered regions of the human proteome and validated HCF1 binding motifs with a position-specific scoring matrix obtained from the binding scores of natural and DMS peptides . ( B ) Heatmap showing the residue in each position of E2F1 and KANSL3 peptides with its effect on WT-corrected binding score based on average DMS of two DHxY peptides, DIDO1 and KANSL3. ( C ) Staining of E. coli cells displaying the indicated peptides with HCF1(Kelch)-EGFP in flow cytometry. Data is the average of two biological replicates, error bars show standard deviation. ( D ) Scatter plot comparing the enrichment scores predicted by the first-order additive model from PSSM scores to the experimentally measured enrichment score, for each variant. The dotted curve is a monotonic I_spline regression fit from to match the predicted and observed values, while accounting for global non-linearities. Variants with residuals that deviate from a residual threshold of 2 standard deviations from the best-fit line are considered epistatic, with points above the fit labelled as positively epistatic, and conversely, negatively epistatic below the spline-fit. ( E ) Comparison of the epistasis effect between DHxY and EHxY-type peptides identified from the scatter plot. Statistical significance between the two groups was assessed using a two-sided Mann–Whitney U test, and the corresponding p-value is shown above the plot. ( F ) Heatmap showing the preference of indicated residues in the ‘x’ position in the DMS experiments in . ( G ) The correlation between residue size and preference score obtained from the DMS experiments in the ‘x’ position of four HBM peptides.

Article Snippet: To test the interaction between recombinant HBM peptides and HCF1, the peptide-GFP proteins were loaded onto ChromoTek GFP-Trap® Magnetic Agarose (Proteintech), followed by incubation with human cell lysate containing endogenous HCF1.

Techniques: Binding Assay, Residue, Staining, Flow Cytometry, Standard Deviation, Variant Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

doi: 10.64898/2026.03.19.712392

Figure Lengend Snippet: ( A ) Plot showing the proteins outcompeted in HCF1 binding by KANSL3 DHxY competitor peptide. Data is from . Mapping of HCF1 binding motifs and the protein interaction network is in Fig. S4A. ( B ) Superimposed DIDO1 DHxY and SMCHD1 DHxxY peptides from peptide-HCF1 complex AlphaFold3 models. ( C ) HCF1(Kelch)-GFP binding of 275 D/EHxxY peptides from the human proteome measured in bacterial surface display. ( D ) Peptide-GFP pulldown from HCT116 cell lysate using recombinant peptides to test binding to HCF1. A representative example of two biological replicates is shown. ( E ) Dose-response curves measuring KANSL3 and IRF1 peptides as competitors to displace a FITC-labeled HCF1 binding peptide from HCF1, measured by fluorescence polarization (n = 2 biological replicates). ( F ) Wild-type-corrected HCF1 binding scores measured in bacterial peptide display with DMS libraries of IRF1 peptide. Below, the sequence logo showing positional preferences for HCF1 binding to IRF1. ( G ) The Gini index (selectivity) showing inequality between residues in a position, calculated from DMS data, and the number of van der Waals contacts of the residue with HCF1 in AlphaFold3 model.

Article Snippet: To test the interaction between recombinant HBM peptides and HCF1, the peptide-GFP proteins were loaded onto ChromoTek GFP-Trap® Magnetic Agarose (Proteintech), followed by incubation with human cell lysate containing endogenous HCF1.

Techniques: Binding Assay, Recombinant, Labeling, Fluorescence, Sequencing, Residue

Journal: bioRxiv

Article Title: HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

doi: 10.64898/2026.03.19.712392

Figure Lengend Snippet: ( A ) The effect of overexpression of different IRF1 variants on MDA-MB-231 cell proliferation. In IRF1-NB, the DHxxY motif is inactivated with Y164A mutation. In IRF1-HB, the DHxxY motif is replaced with a stronger DHxY motif from KANSL3. Data is average from three biological replicates, error bars show standard deviation. ( B ) The effect of different IRF1 variants on cell cycle measured by DNA staining using propidium iodide. Data is average from three biological replicates, error bars show standard deviation. ( C ) RNAseq experiment showing the effect of doxycycline-driven induction of wild-type and mutant IRF1 on gene expression. ( D ) The impact of different IRF1 variant overexpression on interferon response genes and genes involved in TNFα signalling via NF-kB. ( E ) Upregulation of interferon response genes by different IRF1 variants measured in RNAseq. ( F ) Upregulation of TNFα signalling via NF-kB genes by different IRF1 variants measured in RNAseq. ( G ) Model of IRF1 co-regulation by HCF1.

Article Snippet: To test the interaction between recombinant HBM peptides and HCF1, the peptide-GFP proteins were loaded onto ChromoTek GFP-Trap® Magnetic Agarose (Proteintech), followed by incubation with human cell lysate containing endogenous HCF1.

Techniques: Over Expression, Mutagenesis, Standard Deviation, Staining, RNA sequencing, Gene Expression, Variant Assay

Journal: bioRxiv

Article Title: HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

doi: 10.64898/2026.03.19.712392

Figure Lengend Snippet: ( A ) Vulcano plot showing decrease in O-GlcNAc-modification of 39 proteins upon expression of KANSL3 DHxY competitor peptide in HCT116 cells. ( B ) Enrichment of GO terms (Process, Component, and UniProt keywords) among the 39 proteins that are less O-GlcNAc modified in the presence of DHxY competitor peptide in HCT116 cells. ( C ) Scheme illustrating the role of Kelch domain of HCF1 in recruiting glycosylation substrates for OGT. ( D ) Summary model of HCF1 Kelch domain docking interactions. Φ denotes hydrophobic residues.

Article Snippet: To test the interaction between recombinant HBM peptides and HCF1, the peptide-GFP proteins were loaded onto ChromoTek GFP-Trap® Magnetic Agarose (Proteintech), followed by incubation with human cell lysate containing endogenous HCF1.

Techniques: Modification, Expressing, Glycoproteomics