human β1 integrin  (Developmental Studies Hybridoma Bank)

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: E0771 cells have reduced expression of the high-affinity reovirus attachment receptor JAM-A. E0771 and L929 cells were harvested using CellStripper, fixed with 4% paraformaldehyde (PFA), and analyzed by flow cytometry following immunostaining with monoclonal antibodies specific for SNA, JAM-A, or β1 integrin. Representative histograms (left) and corresponding quantification (right) show cell surface expression of ( A ) α2,6-linked sialic acids detected using fluorescently labeled SNA lectin in neuraminidase-treated (+) or untreated (−) cells, ( B ) JAM-A detected using murine JAM-A-specific antibodies (+) compared with isotype (Iso) controls, and ( C ) β1 integrin detected using β1 integrin-specific primary antibodies (+) compared with Iso controls. MFIs were normalized to the corresponding negative controls (neuraminidase-treated or isotype-stained cells) from a representative L929 experiment. Data represent mean ± SD ( n = 3). Statistical significance was determined by the two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism v10.4 (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Expressing, Flow Cytometry, Immunostaining, Bioprocessing, Labeling, Staining

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutations in the sialic acid-binding domain permit high-affinity, receptor-independent attachment by increasing binding to sialic acids at physiological temperature (37°C). ( A ) Representative western blot analysis of outer capsid proteins µ1C and σ3, detected using anti-reovirus polyclonal serum. ( B and C ) L929 ( B ) and E0771 ( C ) cells were treated with PBS or neuraminidase (+neuraminidase) for 1 h at 37°C to deplete cell surface sialic acids. Viruses indicated in the legend were incubated with cells for 1 h at 4°C or 37°C in the presence of NH 4 Cl, washed, and cells processed for flow cytometric analysis using σ3-specific antibodies. MFI reflects the level of cell-associated virus particles. ( D ) Virus-cell association was measured as in panel C , without neuraminidase treatment, using parental U937 cells or U937 cells deficient in sialic acids (U937-Sia - ). ( E ) Flow cytometric detection of α2,6-linked sialic acids on RBCs using fluorescently labeled SNA lectin. Representative histograms (left) show unstained RBCs (light gray), SNA-stained RBCs (red), and SNA-stained H1299 cells (dark gray). n = 3. ( F ) Flow cytometric detection of JAM-A on RBCs using a primary/secondary antibody system specific for hJAM-A. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and E0771+JAM cells (dark gray). n = 3. ( G ) Flow cytometric detection of β1 integrins on RBCs using primary/secondary antibodies specific for human β1 integrin. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and H1299 cells (dark gray). n = 3. ( H ) RBCs were incubated with particle-normalized T3D PL at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C or 37°C. Unbound virions were removed by PBS washes prior to fixation and immunostaining for outer capsid proteins, followed by flow cytometric analysis. n = 3. ( I–K ) RBCs were incubated with particle-normalized T3D PL or variant viruses at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C ( I and J ) or 37°C ( I and K ). Following removal of unbound virions by PBS washes, cells were fixed, immunostained for outer capsid proteins, and analyzed by flow cytometry. Absolute MFI values ( I ) were used to calculate the AUC for each virus across all independent experiments, normalized to T3D PL at the corresponding temperature for each independent experiment ( n = 3–5). ( L ) Levels of σ1 per virion for full-length T3D PLσ1-G196R were assessed by agarose gel electrophoresis (top) and quantitative serial dilution-based western blot analysis using anti-σ3 and anti-µ1 monoclonal antibodies and anti-σ1 tail polyclonal antibodies (middle). Bottom: relative average σ1 per virion calculated relative to T3D PL from five independent virus preparations based on σ1 to (σ3 + µ1) protein ratios determined by western blot analysis. Data represent mean ± SD. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test ( E, J, and K ) or the paired t -test ( F, G, and H ) in GraphPad Prism v10.4. (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Binding Assay, Western Blot, Incubation, Virus, Labeling, Staining, Immunostaining, Variant Assay, Flow Cytometry, Agarose Gel Electrophoresis, Serial Dilution, Bioprocessing

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutational enhancement of σ1-mediated receptor binding and structural basis of sialic acid interaction. ( A ) Model depiction of relative binding strengths deduced from experimental mean AUCs between the sialic acid-binding domain (orange) to sialic acids (dark gray, Sia), the JAM-A-binding domain (circular head of σ1) to JAM-A (black), and the RGD domain (red) to β-integrin (light gray, βInt). Where “~” is indicated, relative binding strength was deduced by subtracting the total binding measured in the JAM-A-deficient condition from the domain-specific binding strength. ( B ) Structural models of the T3D PL σ1 body domain were generated using UCSF ChimeraX (v1.9). Wild-type (bordered) and mutant σ1 structures were created by introducing identified substitutions. Predicted hydrogen bonds with α2,3-linked sialic acid (PDB: 3S6X) were assessed using default cutoffs; a representative G196R rotamer shows novel hydrogen bonds between the arg196 and sialic acid (red arrow). ( C ) Amino acid sequence alignment of the σ1 body domain encompassing the sialic acid-binding pocket (NCBI). Mutations identified through passage (G196R, T193M, and N206H) are annotated alongside known sialic acid-binding residues (N198, R202, and P204).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Binding Assay, Generated, Mutagenesis, Sequencing

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: Proteomics reveals impairment of ECM degradation in PSCs with USP1 knockdown. A , B . USP1 expression was estimated in PSCs treated with different concentration of TGF-β1 (n = 3). C , D . The USP1 expression after lentiviral transfection was detected by real-time PCR ( C ) and western blot ( D ) ( n = 3). E Heap map of differentially expressed proteins (DEPs) in label-free proteomic ( n = 4). F . GO enrichment analysis of DEPs associated with collagen fibers. G . Heat map of collagen protein (n = 4). H . The protein expression of COL1A1, COL1A2, and FN in the PSCs was determined by western blot ( n = 3). I . Immunofluorescence staining of COL1A1 in the PSCs ( n = 3). Bar: 50 μm. J . Immunofluorescence staining of α-SMA. Bar: 50 μm. K . Quantitative analysis of the fluorescence intensity of COL1A1 ( I ) and α-SMA (J) ( n = 3). **, p < 0.01; ***, p < 0.001

Article Snippet: For human TGF-β1 (hTGF-β1) induction, cells were treated with 5 ng/mL hTGF-β1 (HY- P78668 , MCE, New Jersey, USA) for 0, 12, 24, or 48 h. For lentiviral infection, cells were cultured in virus-containing medium for 48 h; subsequently, infected cells were treated with 5 ng/mL hTGF-β1 for 24 h for further analysis.

Techniques: Knockdown, Expressing, Concentration Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: USP1 knockdown promotes ITGB5 ubiquitination-mediated degradation. ( A ) Venn plot showing a total of 2015 proteins bind to USP1 under TGF-β1 stimulation. (B) KEGG pathway analysis and GO enrichment analysis pair of 2015 proteins in ( A ). ( C ) Label-free proteomics combined with IP-LC/MS to analysis the USP1 downstream target. The intersection of differential downregulated expressed protein of label-free proteomics, IP-LC/MS, and the upregulated genes of GSE41418 dataset. ( D ) The ITGB5-associated PPI was established via the String database. E-F. The expression of ITGB5 of the PSCs (E) ( n = 3) and pancreatic tissues ( F ) ( n = 6) was estimated by western blot. G . Co-IP shows the interaction of USP1 and ITGB5 in the PSCs ( n = 3). H . Ubiquitination detection of ITGB5 in the PSCs were measured by Co-IP ( n = 3). **, p < 0.01; ***, p < 0.001.

Article Snippet: For human TGF-β1 (hTGF-β1) induction, cells were treated with 5 ng/mL hTGF-β1 (HY- P78668 , MCE, New Jersey, USA) for 0, 12, 24, or 48 h. For lentiviral infection, cells were cultured in virus-containing medium for 48 h; subsequently, infected cells were treated with 5 ng/mL hTGF-β1 for 24 h for further analysis.

Techniques: Knockdown, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Glycocalyx micro- and nanodomains in cell-cell and cell-matrix interactions revealed by enhanced click chemistry

doi: 10.1038/s41467-026-69242-1

Figure Lengend Snippet: a Single- and multi-channel micrographs (maximum intensity projections) of migrating cells. ROIs: 1) leading edge protrusion, 2) membrane bleb, 3) retraction fiber and 4) collagen contact-free membrane. White arrow, migration direction. Scale bar, 5 µm. b Zoom of leading pseudopod from ( a ). White arrowheads and insets (A, B), β1 clusters outward-segregated from glycocalyx. Scale bar, 2 µm. c Size of 499 β1 clusters from 22 leading edge protrusions (7 cells, 3 independent experiments). d Representative micrographs (from inset A, panel b) of β1-glycocalyx segregation. White arrowhead and line denote β1 cluster and ROI used for outer cluster analysis. Blue line/arrowhead, lateral ROI/boundaries for β1 cluster-adjacent inner zone. Yellow arrowheads, β1 cluster-associated collagen fibers. Collagen channel, Fire pseudocolor. Asterisk, intersection point of both line ROIs. Scale bar, 1 µm. o, outer cluster; i, inner cluster. e Quantification of β1-glycocalyx distance segregation in individual contact to collagen fibril. Magenta/yellow dashed lines, cluster /glycocalyx enrichment middle, determined by maximum β1/glycocalyx levels for outer clusters and corresponding peak in the lateral ROI (inner zone). Blue box, β1 cluster edges, based on the peak-adjacent lateral minima. f , g paired β1 ( f ) and glycocalyx ( g ) enrichment in outer β1 cluster and corresponding lateral membrane zone, normalized to matched membrane region lacking β1 clustering (“nonfocal”). 25 (cell body) and 38 (inner-outer matched) line ROIs from 9 cells of 3 independent experiments. Wilcoxon Rank-Sum test with Bonferroni correction (ε 2 = 0.25 ( f ) and ε 2 = 0.54 ( g ), large effect size). h Segregation distance of β1 and glycocalyx in outer β1 clusters. Data show 25 individual perpendicular membrane regions and 38 focal outward clusters from 11 cells of 3 independent experiments. Wilcoxon Rank-Sum test (ε 2 = 0.56, large effect size). i Correlation of local glycocalyx density and β1 enrichment in outward β1 clusters (R-squared = −0.02, adjusted p -value = 1). Data replotted from ( h ). Line, logarithmic fitting curve with 95% confidence interval (ribbon). All data derive from the same 3 independent experiments. Cells (all panels): MV3. Boxplots: middle-line, median; outlines, 1 st -3 rd quantiles; whiskers, quantiles ±1.5x interquantile range. ROI region of interest. β1, β1 integrin. Source data are provided as a Source Data file.

Article Snippet: For β1 integrin staining, collagen-embedded cells were incubated in blocking buffer (1 % bovine serum albumin, Sigma-Aldrich, Cat# A9647; 10 % normal goat serum, Thermo Fisher Scientific, Cat# 10000 C; PBS, 1 h, 20 °C), incubated with a mixture of two mouse anti-human β1 integrin antibodies (clone K20, Novus Biochemicals, NBP2-52708; clone 4B4LDC9LDH8, Beckman Coulter, 6603113; both 10 ug/mL in 50 μl, blocking buffer, 24 h, 4 °C, mild agitation), washed 3 times (blocking buffer, 15 min, 4 °C) and incubated with secondary antibody mouse IgG (H + L) highly cross-adsorbed AlexaFluor647 (2 μg/ml in 50 μl, Thermo Fisher Scientific, Cat# A21236, 24 h, 4 °C), 1 μg/mL DAPI (Merck, Cat# D9542), and when non-fluorescent collagen was used, with 2U/ml Phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A12380) (washed again 3x, PBS, 15 min, 4 °C).

Techniques: Membrane, Migration

Journal: Nature Communications

Article Title: Glycocalyx micro- and nanodomains in cell-cell and cell-matrix interactions revealed by enhanced click chemistry

doi: 10.1038/s41467-026-69242-1

Figure Lengend Snippet: a – f Glycocalyx/β1 fluorescence in leading pseudopod ( a ) and quantification of single ( b ) and multiple ( c , d ) pseudopods normalized by average non-contacting membrane fluorescence. Multichannel and single-channel micrographs from 3-slice maximum-intensity projections from Fig. (region 1) showing glycocalyx along each pseudopod ( d ), vs. β1 enrichment ( e ) or per glycocalyx intensity category ( f ). Line in ( a ), quantification line in ( b ), with colors in ( a ) matching shades in ( b ). Scale bar, 2 µm. Dashed/solid vertical lines, β1 cluster peaks/edges, respectively. Datapoints ( c – f ): 449 β1 clusters from 22 protrusions, 7 cells. Black lines, linear ( d ) and logarithmic ( e ) fit ± 95% CI (ribbon). Calculation ( e , f ), see Supplementary Fig. . Categorized glycocalyx in 3 content groups based on total cluster number. g – i Glycocalyx/β1 distributions in blebs using 3-slice maximum-intensity projections ( g ; indicated in Figs. a- , post-rotation), fluorescence intensity in single bleb ( h ) and multiple blebs ( i ). Line subsegment colors in ( g ), shaded areas in ( h ). Yellow arrowhead, bleb apex. Pseudocolor: Fire-LUT. Scale bar, 2 µm. i Mean glycocalyx intensity normalized to mean collagen-contact-free membrane region; 32 blebs, 12 cells. j Glycocalyx vs. β1 fluorescence in blebs and paired bleb apexes (lines). Datapoints replotted from ( i ). k – m Glycocalyx/β1 fluorescence micrograph (3-slice maximum-intensity projections) ( k ; from Figs. a– ) and quantification along single ( l ) and multiple retraction fibers compartments corrected for collagen-contact-free fluorescence ( m ) and along relative fiber length ( n ). Line in ( k ), quantification line matching ( l ). In ( l ): Solid/dashed lines, cluster edges/centers, respectively. Datapoints ( m ): 328 clusters from 13 retraction fibers, 5 cells. Data in ( d , n ): clusters (dots) on the same protrusion (connected lines distinguished by colors). Line, linear fit ± 95% CI. R values, adjusted coefficient of determination. P.adj, adjusted p-value (all panels). All panels: Kruskall-Wallis test with Bonferroni correction (ε = 0.06 ( f ), indicates moderate effect size; ε = 0.39 ( c ), ε = 0.19 ( i ) and ε = 0.39 ( m ) indicate high effect sizes). β1, β1 integrin. Data present the same 3 independent experiments as Fig. . Boxplots: middle-line, median; outlines, 1 st -3 rd quantiles; whiskers, quantiles ±1.5x interquantile range. CI confidence interval. Source data are provided as a Source Data file.

Article Snippet: For β1 integrin staining, collagen-embedded cells were incubated in blocking buffer (1 % bovine serum albumin, Sigma-Aldrich, Cat# A9647; 10 % normal goat serum, Thermo Fisher Scientific, Cat# 10000 C; PBS, 1 h, 20 °C), incubated with a mixture of two mouse anti-human β1 integrin antibodies (clone K20, Novus Biochemicals, NBP2-52708; clone 4B4LDC9LDH8, Beckman Coulter, 6603113; both 10 ug/mL in 50 μl, blocking buffer, 24 h, 4 °C, mild agitation), washed 3 times (blocking buffer, 15 min, 4 °C) and incubated with secondary antibody mouse IgG (H + L) highly cross-adsorbed AlexaFluor647 (2 μg/ml in 50 μl, Thermo Fisher Scientific, Cat# A21236, 24 h, 4 °C), 1 μg/mL DAPI (Merck, Cat# D9542), and when non-fluorescent collagen was used, with 2U/ml Phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A12380) (washed again 3x, PBS, 15 min, 4 °C).

Techniques: Fluorescence, Membrane

Journal: Nature Communications

Article Title: Glycocalyx micro- and nanodomains in cell-cell and cell-matrix interactions revealed by enhanced click chemistry

doi: 10.1038/s41467-026-69242-1

Figure Lengend Snippet: a Nanoscale segregation of glycocalyx from β1 integrin cluster in a perpendicular direction. The two-compartment zone consists of an outer β1 integrin cluster with low glycocalyx content, segregating perpendicularly from a glycocalyx-rich region at the cell body with variable β1 integrin enrichment, yet a lack of glycocalyx segregation at the base of this interaction. The outer β1 integrin outer cluster interacts with fibrillar collagen and is connected to the actin cytoskeleton, consistent with a glycocalyx-deficient nanoprotrusion. b I) Micron-scale glycocalyx depletion across a leading edge protrusion from the base towards the apical direction. Dashed rectangle, inset II), which illustrates that glycocalyx-depleted zones of the tip of the leading edge protrusion form a zone of high-integrin clustering sensitivity. c Micron-scale glycocalyx depletion in blebs towards the bleb apex. d Micron-scale glycocalyx depletion towards the tip of retraction fibers. e Micron-scale glycocalyx underrepresentation in cell-cell contacts and gradient-like redistribution out of cell-cell contact along single-cell membrane segments and interconnecting transition zone. In all panels, solid arrows indicate migration direction, and dashed arrows indicate glycocalyx depletion direction.

Article Snippet: For β1 integrin staining, collagen-embedded cells were incubated in blocking buffer (1 % bovine serum albumin, Sigma-Aldrich, Cat# A9647; 10 % normal goat serum, Thermo Fisher Scientific, Cat# 10000 C; PBS, 1 h, 20 °C), incubated with a mixture of two mouse anti-human β1 integrin antibodies (clone K20, Novus Biochemicals, NBP2-52708; clone 4B4LDC9LDH8, Beckman Coulter, 6603113; both 10 ug/mL in 50 μl, blocking buffer, 24 h, 4 °C, mild agitation), washed 3 times (blocking buffer, 15 min, 4 °C) and incubated with secondary antibody mouse IgG (H + L) highly cross-adsorbed AlexaFluor647 (2 μg/ml in 50 μl, Thermo Fisher Scientific, Cat# A21236, 24 h, 4 °C), 1 μg/mL DAPI (Merck, Cat# D9542), and when non-fluorescent collagen was used, with 2U/ml Phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A12380) (washed again 3x, PBS, 15 min, 4 °C).

Techniques: Single Cell, Membrane, Migration