rabbit anti mouse integrin α3  (R&D Systems)

Journal: bioRxiv

Article Title: Orthogonal Force Balance Between Contractility and Shear Stress Governs Podocyte Dynamics

doi: 10.64898/2026.01.22.701159

Figure Lengend Snippet: a, Model-predicted integrin stability as a function of location from foot process periphery to center under varying shear stress conditions. Low stress (blue) shows minimal peripheral preference; increasing stress (orange to red) drives progressive peripheral accumulation with central depletion. b, Schematic of predicted integrin redistribution under mechanical stress. Integrins accumulate at foot process peripheries (green) as stress increases, with potential shape changes and edge detachment under excessive loading. c, Airyscan super-resolution imaging validates predicted pattern in healthy mouse glomerulus. Integrin α3 (red) accumulates in gaps between synaptopodin-marked foot processes (green), with nephrin marking slit diaphragms (blue). Scale bar: 1 μm. d, Relative fluorescence intensity (RFI) plot along indicated line in panel c shows integrin α3 peaks (red) localized between synaptopodin peaks (green), confirming peripheral accumulation pattern. e Expansion microscopy (4× expansion) enables single foot process resolution. Podocalyxin (membrane marker, magenta) encapsulates central synaptopodin (green), with integrin α3 (red) co-localizing at periphery. Scale bar: 1 μm. f, Quantitative analysis of straightened foot processes. Integrated RFI plot from all pixels surrounding foot processes shows central synaptopodin peak flanked by two peaks in both podocalyxin and integrin α3 channels, definitively confirming peripheral integrin localization matching model predictions.

Article Snippet: The first and second antibodies used included: Guinea-pig anti-mouse synaptopodin (ARP, 03-GP94-N, Waltham, MA, USA); Rabbit anti-mouse integrin-α3 (BiCell, 10003, St. Louis, MO, USA); Goat anti-mouse nephrin (R&D System, AF3159, Minneapolis, MN, USA); Goat anti-mouse podocalyxin (R&D System, AF1556, Minneapolis, MN, USA); Alexa fluor-488 Donkey anti-guinea-pig secondary (Jackson ImmunoResearch, 706-545-148, West Grove, PA, USA); Alexa fluor-594 Donkey anti-rabbit secondary (Jackson ImmunoResearch, 711-585-152, West Grove, PA, USA); Dylight-405 Donkey anti-rabbit secondary(Jackson ImmunoResearch, 711-475-152, West Grove, PA, USA); and Alexa fluor-647 Donkey anti-goat secondary (Jackson ImmunoResearch, 705-605-003, West Grove, PA, USA).

Techniques: Shear, Imaging, Fluorescence, Microscopy, Membrane, Marker

Journal: bioRxiv

Article Title: Orthogonal Force Balance Between Contractility and Shear Stress Governs Podocyte Dynamics

doi: 10.64898/2026.01.22.701159

Figure Lengend Snippet: a, Airyscan imaging reveals integrin α3 localization at foot process peripheries in both low BP and high BP mice 60 minutes post-blebbistatin. Synaptopodin marks central actin cables (green), integrin α3 shown in red. Scale bar: 1 μm. b, Relative fluorescence intensity plots along indicated lines in panel c demonstrate enhanced integrin accumulation in high BP mice. Greater peak-to-valley intensity differences in high BP samples indicate increased peripheral concentration under elevated shear stress. c, Expansion microscopy reveals foot process boundaries in both low and high BP mice. Podocalyxin staining (magenta) clearly identifies peripheries, with integrin α3 (red) accumulation partially lost in some low BP samples. Scale bar: 1 μm. d, Integrated RFI plots from straightened foot processes show differential integrin distribution. While podocalyxin maintains two peaks surrounding central synaptopodin in both groups, integrin α3 shows widened distribution in low GFR samples versus significant peripheral accumulation in high GFR group, confirming stress-dependent redistribution. e, Airyscan imaging of human kidney samples reveals conserved integrin localization patterns. In healthy human glomerulus (left), integrin α3 (red) localizes at foot process peripheries around synaptopodin-marked central actin (green). In minimal change disease (right), integrin α3 accumulates precisely between effaced foot processes, with sarcomere-like structures (SLSs) visible as discontinuous synaptopodin signals (arrows). Scale bar: 1 μm. f, Relative fluorescence intensity plots from human samples demonstrate peripheral integrin accumulation away from central synaptopodin signals in both healthy and diseased tissue, confirming conservation of the stress-responsive redistribution mechanism across species.

Article Snippet: The first and second antibodies used included: Guinea-pig anti-mouse synaptopodin (ARP, 03-GP94-N, Waltham, MA, USA); Rabbit anti-mouse integrin-α3 (BiCell, 10003, St. Louis, MO, USA); Goat anti-mouse nephrin (R&D System, AF3159, Minneapolis, MN, USA); Goat anti-mouse podocalyxin (R&D System, AF1556, Minneapolis, MN, USA); Alexa fluor-488 Donkey anti-guinea-pig secondary (Jackson ImmunoResearch, 706-545-148, West Grove, PA, USA); Alexa fluor-594 Donkey anti-rabbit secondary (Jackson ImmunoResearch, 711-585-152, West Grove, PA, USA); Dylight-405 Donkey anti-rabbit secondary(Jackson ImmunoResearch, 711-475-152, West Grove, PA, USA); and Alexa fluor-647 Donkey anti-goat secondary (Jackson ImmunoResearch, 705-605-003, West Grove, PA, USA).

Techniques: Imaging, Fluorescence, Concentration Assay, Shear, Microscopy, Staining

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 negatively regulates ITGA3 and EphA2 expression. (A) Violin plots of ITGA3 or EphA2 expression levels in subpopulations are shown using the same single‐cell RNA‐seq data with Figure . The subpopulations with higher invasive scores was marked by red line. (B) A t‐SNE analysis of single‐cell RNA‐seq data from GSE134432 . Violin plots of SOX10, ITGA3, or EphA2 expression levels in each cluster are shown. (C) Human melanoma cells were transfected with siCNTL or siSOX10 (#9 or #10) and whole‐cell lysates 3 days after siRNA transfection were subjected to Western blotting. (D) A2058 cells were infected with shRNA for control (shSCR) or SOX10 (shSOX10). After puromycin selection, whole‐cell lysates were subjected to Western blotting. (E) A2058 cells were transfected with siCNTL, siSOX10 (#9 or #10), or siMITF (#1 or #2). Three days after siRNA transfection, whole‐cell lysates were subjected to Western blotting. Other conditions were similar to those in Figure .

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, RNA Sequencing, Transfection, Western Blot, Infection, shRNA, Control, Selection

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 regulates melanoma adhesion and motility through ITGA3 and EphA2, respectively. (A) A2058/Luc cells were transfected with siCNTL or siSOX10 (#9 or #10). Three days after transfection, cells (1.25 × 10 3 cells/well) were added to non‐ (−) or laminin‐332‐coated wells (LN‐332). The intensity of luminescence was measured by an in vivo imaging system. Data are the means ± SD with each data point. * p < 0.01 versus siCNTL‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. (B, C) A2058/Luc or A2058 cells were transfected with siCNTL, siSOX10 #10, siITGA3 (#1 or #2), or siEphA2 (#1 or #2) for 3 days. A2058/Luc cells were subjected to an adhesion assay (left) and A2058 cells to a random motility assay using the time‐lapse imaging system (center), while whole‐cell lysates were subjected to Western blotting (right). Data are the means ± SD with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. (D) Human melanoma cells were transfected with siCNTL or siSOX10 (#9 or #10). Three days after transfection, cells were subjected to the transwell‐chamber migration assay. After hematoxylin and eosin staining, the number of migrated cells was counted under a microscope. Data are the means ± SD with each data point. * p < 0.01 versus siCNTL‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test in each cell line. (E) A2058 cells were transfected with siCNTL, siSOX10#10, siITGA3 (#1), or siEphA2 (#2) for 3 days. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. Other conditions were similar to those in (D).

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, In Vivo Imaging, Cell Adhesion Assay, Motility Assay, Imaging, Western Blot, Migration, Staining, Microscopy

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 regulates EphA2 and ITGA3 through the transcription factor IRF1. (A) Genes that positively correlated with EphA2 or ITGA3 are shown in melanoma from the CCLE dataset. The negative correlation of SOX10 with EphA2 and ITGA3 is shown in the upper panel. (B–F) A2058 cells were transfected with siCNTL, siSOX10 #10, or siIRF1 (#1 or #2). Three days after transfection, whole‐cell lysates were subjected to Western blotting (B). Three days after transfection, transfected cells were subjected to an adhesion assay (C), random motility assay (D), transwell‐migration assay (E), or experimental lung metastasis assay (F). Data show the mean ± SD (C–E) or ± SEM (F) with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Cell Adhesion Assay, Motility Assay, Transwell Migration Assay

Journal: Cancer Science

Article Title: SOX10 Regulates Melanoma Metastasis Through the IRF1 ‐ ITGA3 / EphA2 ‐ FAK Pathway

doi: 10.1111/cas.70173

Figure Lengend Snippet: SOX10 regulates melanoma metastasis to laminin‐332 through the ITGA3/EphA2‐FAK axis. (A) A gene set enrichment analysis was performed by KEGG gene sets using bulk RNA‐seq data after the knockdown of SOX10 from Figure . The enrichment plot of KEGG_Focal_Adhesion is shown. (B, C) A2058 cells were transfected with siCNTL, siSOX10 #10, siIRF1 (#1 or #2), siEphA2 #2, or siITGA3 #1. Three days after transfection, whole‐cell lysates were subjected to Western blotting. (D) A2058 cells were transfected with siCNTL or siSOX10 #10. Three days after transfection, transfected cells were treated with defactinib (0.1 or 1 μM) for 2 h and then subjected to a transwell‐migration assay. Defactinib was added to both the upper and lower chambers during the transwell‐migration assay (left panel). After 2 h of exposure to defactinib, whole‐cell lysates were subjected to Western blotting (right panel). Data show the mean ± SD with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test. (E) A2058/Luc cells were transfected with siCNTL or siSOX10 #10. Three days after transfection, transfected cells were treated with defactinib (1 μM) for 8 h and then subjected to the experimental lung metastasis assay. Data are shown as the mean ± SEM ( n = 5) with each data point. * p < 0.01 versus siSOX10‐transfected cells by a one‐way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: The primary antibodies used were MITF (C‐5) from Dr. Fisher, D.E. (MGH), AXL (#8661), EphA2 (#6997), Phospho‐EphA2 (Ser‐897; #6347), IRF1 (#8478), FAK (#3285), and Phospho‐FAK (Tyr‐397; #8556) from Cell Signaling Technology (Beverly, MA, USA), and ITGA3 (sc‐374242), ITGB1 (sc‐374429), SOX10 (sc‐514302), and β‐actin (sc‐47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: RNA Sequencing, Knockdown, Transfection, Western Blot, Transwell Migration Assay

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A Quantifications of HMEC-1 cell migration (left) and tube formation (right) induced by recombinant Gal-3 under an insulin-resistant state (treated with serum-free medium containing 100 nM insulin for 24 h) were shown ( n = 4 biological replicates each group). -, normal state; +, insulin-resistant state. B RT-qPCR analysis of VEGFA , FGF2 , and HGF in HMEC-1 cells treated with the indicated concentration of recombinant Gal-3 for 12 h. Relative expression levels were normalized to ACTB ( n = 3 biological replicates each group). C Heatmap of proteomic abundance (normalized using Z-score) of the top 5 Gal-3-interacting proteins in skin endothelial cells from healthy donors (dataset PXD019909, ProteomeXchange). D HMEC-1 cell migration (left) ( n = 4 biological replicates) and tube formation (right) ( n = 3 biological replicates) induced by recombinant Gal-3 with knockdown of Catenin α-1 or integrin β1. E GST pull-down assays. HMEC-1 cell lysate was incubated with GST or GST-Gal-3 and pulled down with GS beads (left panel); cells were treated with GST or GST-Gal-3 at 4 °C for 1 h, cross-linked, lysed and pulled down with GS beads (right panel). GST served as a negative control. Immunoblot analysis of integrin β1 was shown. F Recombinant Gal-3-induced migration of HMEC-1 cells with integrin β1-targeting shRNAs or non-targeting shRNA (shscr) ( n = 3 biological replicates each group). G Recombinant Gal-3-induced migration of HMEC-1 cells incubated with integrin β1 functional blocking antibody (TDM29, 10 µg/mL) or IgG control (left). Quantifications were shown ( n = 3 biological replicates each group). Scale bar, 500 μm. H Schematic diagram of the α subunit partnering with the integrin β1 subunit created in BioRender. Chen, S. (2025) https://BioRender.com/p10vue6 . Among the 12 α subunits, α3, α5, and α6 subunits were detected by the mass spectrometry analysis in the His-Gal-3 immunoprecipitation assay performed in HMEC-1 cells (see Supplementary Fig. ). I GST pull-down assays. HMEC-1 cell lysate was incubated with GST or GST-Gal-3 and pulled down with GS beads (left panel); cells were treated with GST or GST-Gal-3 at 4 °C for 1 h, cross-linked, lysed and pulled down with GS beads (right panel). GST served as a negative control. Immunoblot analysis of integrin α5, integrin α6 and integrin α3 was shown. J Recombinant Gal-3 induced migration of HMEC-1 cells incubated with integrin α5 functional blocking antibody P1D6 (10 µg/mL) ( n = 3 biological replicates each group). K Recombinant Gal-3-induced migration of HMEC-1 cells that were pre-incubated with integrin α5β1 antagonist ATN-161 (100 nM) for 48 h ( n = 5 biological replicates each group). L , Immunoblot analysis of the phosphorylation of integrin β1 (p-integrin β1, Ser783) in HMEC-1 cells that were incubated with recombinant Gal-3 (10 μg/mL). Relative expression levels were normalized to integrin β1, and quantifications were shown below the blots. M Immunoblot analysis and quantifications of p-integrin β1 in wounds of diabetic mice that i.c . injected with OE-Gal-3 adenovirus or Veh. Relative expression levels were normalized to integrin β1 ( n = 3 biological replicates each group). All statistical data points are represented as means ± SEM. P values were determined by unpaired two-tailed Student’s t -test ( A , B , D , F , G , J , K , M ) or one-way ANOVA with Fisher’s LSD post hoc test ( B , D , F ). Error bars mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: After blocking with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20), blots were incubated overnight at 4°C with the following primary antibodies: CD31 (1:1000, ab281583, Abcam, USA), β-Actin (1:1000, 612657, BD Biosciences, USA), Gal-3 (1:1000, ab76245, Abcam, USA), collagen I (1:1000, GB114197 , Servicebio, China), collagen III (1:1000, EPR17673 , Abcam, USA), AKT (1:1000, 9272 s, cell signaling technology, USA), p-AKT (Ser473) (1:1000, 9271 s, cell signaling technology, USA), FoxO1 (1:1000, 18592-1-AP, Proteintech, China), integrin β1 (1:1000, 610467, BD Biosciences, USA), integrin α3 (1:1000, 66070, Proteintech, China), integrin α5 (1:1000, sc-10729, Santa Cruz, USA), integrin α6 (1:1000, ab181551, Abcam, USA), p-integrin β1 (Y783) (1:1000, ab62337, Abcam, USA), VEGFR2 (1:1000, 2479S, Cell signaling, USA), p-VEGFR2 (Tyr1175) (1:1000, 2478S, Cell signaling technology, USA), EGFR (1:1000, 4267S, Cell signaling, USA), p-EGFR (Tyr1068) (1:1000, 3777S, Cell signaling technology, USA), integrin αv(1:1000, 27096-1-AP, Proteintech, China), FAK (1:1000, 12636-1-AP, Proteintech China), p-FAK (Y397) (1:1000, 611806, BD Biosciences, USA), CD146 (1:1000, 611208, BD Biosciences, USA), GST (1:1000, KM8005, Sungene Biotech, China), His-tag (1:1000, RM1001, Beijing Ray Antibody Biotech, China), α-Tubulin (1:1000, F0063, Selleck, USA).

Techniques: Migration, Recombinant, Quantitative RT-PCR, Concentration Assay, Expressing, Knockdown, Incubation, Negative Control, Western Blot, shRNA, Functional Assay, Blocking Assay, Control, Mass Spectrometry, Immunoprecipitation, Phospho-proteomics, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A – E STZ-induced diabetic rats were i.c . injected with lgals3 adenovirus or control virus (Veh) after wounding, followed by treatment with integrin β1 functional blocking antibody (Anti-β1) or IgG control (IgG). A Representative images of the wounds (left) and percentage of wound closure (right) ( n = 5). B H&E staining of healed wounds. The distance between the first and second yellow dotted lines represents epidermis thickness (red arrows), and the distance between the second and third yellow dotted lines represents granulation thickness (black arrows). Quantifications of the epidermis and granulation thickness were shown on the lower ( n = 5). Scale bar, 500 µm. C Picrosirius red staining showing COL1 and COL3 in healed wound under polarized light. Quantifications of COL1 area percentage and total COL1 and COL3 area were shown on the right ( n = 5). Scale bar, 50 µm. D Immunohistochemical staining of CD31 that marked microvessels (black arrows) in healed wounds. Quantifications of microvessel count per field were shown on the right ( n = 5). Scale bar, 50 µm. E Immunoblot analysis and quantifications of CD31 in healed wounds ( n = 5). F , G STZ-induced diabetic rats were i.c . injected with shRNA targeting integrin α5 (shα5) or non-targeting shRNA (shscr) 2 weeks before wounding, following i.c . injected with recombinant lgals3 adenovirus (OE-Gal-3) or control virus (Veh). Normal rats (Normal) were set as negative control. F COL1 and COL3 in picrosirius red staining in healed wounds. Quantifications of COL1 area percentage and total COL1 and COL3 area were shown ( n = 5). Scale bar, 50 µm. G Immunohistochemical staining of CD31 (black arrows) in healed wounds. Quantifications of microvessel count per field were shown on the right ( n = 5). Scale bar, 50 µm. H Recombinant Gal-3-induced migration of HMEC-1 cells treated with FAK inhibitor (25 μM), Src-inhibitor (1 μM) or IKK inhibitor (0.5 μM) for 48 h. (Veh, n = 5 biological replicates; FAK inhibitor, n = 3 biological replicates; Src-inhibitor, n = 5 biological replicates; IKK inhibitor, n = 5 biological replicates). I Immunoblot analysis of the phosphorylation of FAK (p-FAK, Y397) in HMEC-1 cells treated with recombinant Gal-3 (10 µg/mL). p-FAK levels were normalized to FAK. Quantifications were shown below the blots. J Recombinant Gal-3-induced tube formation of HMEC-1 cells treated with FAK inhibitor (FAKi, 25 μM), ( n = 4 biological replicates). K Immunoblot analysis (upper) and quantifications (lower) of p-FAK in HMEC-1 cells treated with si-integrin β1 (si-β1) or negative control. p-FAK levels were normalized to FAK ( n = 3 biological replicates). L , M HFD/STZ mice were i.c . injected with recombinant lgals3 adenovirus (OE-Gal-3) or control virus (Veh) after wounding, followed by treatment with FAKi (OE-Gal-3+ FAKi, 15 µM, 100 µL/mouse) or vehicle once every two days. L COL1 and COL3 in picrosirius red staining in healed wounds (left). Quantifications of COL1 area percentage and total COL1 and COL3 area (right). Scale bar, 50 µm. M Immunohistochemical staining and quantifications of CD31 (black arrows) in healed wounds. ( n = 3, two sections per mouse). Scale bar, 50 µm. All statistical data are presented as means ± SEM. P values were determined by unpaired two-tailed Student’s t -test ( A – H , J – M ) or one-way ANOVA with Fisher’s LSD post hoc test ( H ). Error bars mean ± SEM of each group. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: After blocking with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20), blots were incubated overnight at 4°C with the following primary antibodies: CD31 (1:1000, ab281583, Abcam, USA), β-Actin (1:1000, 612657, BD Biosciences, USA), Gal-3 (1:1000, ab76245, Abcam, USA), collagen I (1:1000, GB114197 , Servicebio, China), collagen III (1:1000, EPR17673 , Abcam, USA), AKT (1:1000, 9272 s, cell signaling technology, USA), p-AKT (Ser473) (1:1000, 9271 s, cell signaling technology, USA), FoxO1 (1:1000, 18592-1-AP, Proteintech, China), integrin β1 (1:1000, 610467, BD Biosciences, USA), integrin α3 (1:1000, 66070, Proteintech, China), integrin α5 (1:1000, sc-10729, Santa Cruz, USA), integrin α6 (1:1000, ab181551, Abcam, USA), p-integrin β1 (Y783) (1:1000, ab62337, Abcam, USA), VEGFR2 (1:1000, 2479S, Cell signaling, USA), p-VEGFR2 (Tyr1175) (1:1000, 2478S, Cell signaling technology, USA), EGFR (1:1000, 4267S, Cell signaling, USA), p-EGFR (Tyr1068) (1:1000, 3777S, Cell signaling technology, USA), integrin αv(1:1000, 27096-1-AP, Proteintech, China), FAK (1:1000, 12636-1-AP, Proteintech China), p-FAK (Y397) (1:1000, 611806, BD Biosciences, USA), CD146 (1:1000, 611208, BD Biosciences, USA), GST (1:1000, KM8005, Sungene Biotech, China), His-tag (1:1000, RM1001, Beijing Ray Antibody Biotech, China), α-Tubulin (1:1000, F0063, Selleck, USA).

Techniques: Injection, Control, Virus, Functional Assay, Blocking Assay, Staining, Immunohistochemical staining, Western Blot, shRNA, Recombinant, Negative Control, Migration, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A Confocal microscopy of integrin α5 segregation in HMEC-1 cells treated with recombinant Gal-3 (1.65 µM) plus lactose (10 mM). An enlarged view of the boxed region shows clusters on the cell membrane (red arrows). Quantifications of cluster number per cell (left to right, n = 5, 6, 5 fields; total number of cells were 50-90 in each group). Scale bar, 20 µm. B , C Condensates formed with the GFP-Gal-3 (80 µM), GFP-Gal-3 (80 µM) + integrin β1 (400 nM) mixture, GFP-Gal-3 (80 µM) + integrin β1 (400 nM) mixture plus lactose (20 mM), and GFP-Gal-3 (80 µM) + CD146 (400 nM) mixture in PBS (pH 7.4), respectively. B Fluorescence microscopy images of the condensates (red arrows) and quantifications of condensates’ number and diameter in each microscope field ( n = 5 biological replicates). Scale bar, 10 µm. C Solution turbidity for the indicated mixtures measurements by UV-vis spectrophotometry ( n = 3 biological replicates each group). D , E Condensates formed with the GFP-Gal-3 (80 µM), GFP-Gal-3 (80 µM) + integrin α5β1 (400 nM) mixture, GFP-Gal-3 (80 µM) + integrin α5β1 (400 nM) mixture plus lactose (20 mM), and GFP-Gal-3 (80 µM) + CD146 (400 nM) mixture in PBS (pH 7.4). D Fluorescence microscopy images of the condensates and quantifications of condensates’ number, and diameter of each microscope field were shown ( n = 5 biological replicates). Scale bar, 10 µm. E Solution turbidity for the mixtures (left to right, n = 3, 4, 3, 3 biological replicates). F Solution turbidity for the mixtures formed with Gal-3 (0 µM, 10 µM, 20 μM, 40 µM, 80 µM and 100 µM) and integrin α5β1 (400 nM) ( n = 3 biological replicates). G Fluorescence Recovery After Photobleaching (FRAP) analysis of droplets formed with GFP-Gal-3 (80 µM) and integrin β1 (400 nM), integrin α5β1 (400 nM) or CD146 (400 nM), respectively. Representative confocal microscopy images (left) and normalized fluorescence intensity (right) after bleaching were shown ( n = 5, 4, 5 independent measurements, respectively). H Condensates formed with the GFP-Gal-3 (80 µM) and integrin α5β1 (400 nM) in PBS (pH 7.4) had their N-glycans removed by PNGase. Fluorescence microscopy images of the condensates and quantifications of condensates’ number, and diameter of each microscope field ( n = 4 biological replicates). Scale bar, 10 µm. I Confocal microscopy of Gal-3 (3.3 µM, containing 0.8 µM GFP-Gal-3 and 2.5 µM Gal-3) induced condensates in CHO-K1 cells expressing the mCherry-integrin α5, with or without 1, 6-hexanediol (1, 6-HD) (1.5%, 2 min). An enlarged view of the boxed region was shown on the right, with cross-sectional fluorescence intensity profiles along the white dotted line in histograms demonstrating the correlation between the two signals. Quantifications of the size and the number of the condensates per cell were shown (upper, n = 5, 5, 5 fields; lower, n = 5, 5, 4 fields, total number of cells were 60-70 in each group). Scale bar, 20 μm. J FRAP measurements. The co-localized Gal-3/integrin α5 condensates in living cells were randomly selected for bleaching (upper panel). Enlarged views of the boxed region were shown. Representative confocal microscopy images (middle panel) and normalized fluorescence intensity (lower panel) after bleaching ( n = 5 independent measurements). Scale bar, 10 μm. K Immunoblot analysis and quantifications of the phosphorylation of FAK (p-FAK, Y397) in HMEC-1 cells treated with Gal-3 (0.33 µM, 15 min) together with PBS, lactose (4 mM), or 1, 6-HD (1.5%, 2 min) in the indicated group ( n = 6, 5, 6, 6 biological replicates). L Confocal microscopy of Gal-3 (3.3 µM, containing 0.8 µM GFP-Gal-3 and 2.5 µM Gal-3) induced condensates in CHO-K1 cells co-expressing mCherry-integrin α5 and mTagBFP2-CD146 treated with lactose (10 mM), sucrose (10 mM) or 1, 6-HD (1.5%, 2 min). Enlarged views of the boxed region were shown with corresponding cross-sectional fluorescence intensity profiles along the white dotted line in histograms demonstrating the correlation between the three signals. Quantifications of the size and number of the condensates per cell were shown ( n = 5 fields, total number of cells was 40–80 in each group). Scale bar, 20 μm. M Recombinant Gal-3-induced tube formation in HMEC-1 cells treated with siRNA targeting integrin β1 or CD146 ( n = 3 biological replicates). All statistical data are presented as means ± SEM. P values were determined by unpaired two-tailed Student’s t test ( A , H , M ), one-way ANOVA with Fisher’s LSD post hoc test ( C , E , F , K ), two-sided Mann-Whitney U test ( I , L ) or Kruskal–Wallis test with Dunn’s post hoc test ( B , D ). Error bars mean ± SEM of each group. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: After blocking with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20), blots were incubated overnight at 4°C with the following primary antibodies: CD31 (1:1000, ab281583, Abcam, USA), β-Actin (1:1000, 612657, BD Biosciences, USA), Gal-3 (1:1000, ab76245, Abcam, USA), collagen I (1:1000, GB114197 , Servicebio, China), collagen III (1:1000, EPR17673 , Abcam, USA), AKT (1:1000, 9272 s, cell signaling technology, USA), p-AKT (Ser473) (1:1000, 9271 s, cell signaling technology, USA), FoxO1 (1:1000, 18592-1-AP, Proteintech, China), integrin β1 (1:1000, 610467, BD Biosciences, USA), integrin α3 (1:1000, 66070, Proteintech, China), integrin α5 (1:1000, sc-10729, Santa Cruz, USA), integrin α6 (1:1000, ab181551, Abcam, USA), p-integrin β1 (Y783) (1:1000, ab62337, Abcam, USA), VEGFR2 (1:1000, 2479S, Cell signaling, USA), p-VEGFR2 (Tyr1175) (1:1000, 2478S, Cell signaling technology, USA), EGFR (1:1000, 4267S, Cell signaling, USA), p-EGFR (Tyr1068) (1:1000, 3777S, Cell signaling technology, USA), integrin αv(1:1000, 27096-1-AP, Proteintech, China), FAK (1:1000, 12636-1-AP, Proteintech China), p-FAK (Y397) (1:1000, 611806, BD Biosciences, USA), CD146 (1:1000, 611208, BD Biosciences, USA), GST (1:1000, KM8005, Sungene Biotech, China), His-tag (1:1000, RM1001, Beijing Ray Antibody Biotech, China), α-Tubulin (1:1000, F0063, Selleck, USA).

Techniques: Confocal Microscopy, Recombinant, Membrane, Fluorescence, Microscopy, Spectrophotometry, Expressing, Western Blot, Phospho-proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A Tube formation induced by recombinant Gal-3 in HUVECs with diabetic or non-diabetic patient serum (7.5%, v/v) (Non-DM, n = 5; DM, n = 6 biological replicates). B Recombinant Gal-3-induced tube formation in HUVECs treated with different concentrations of BSA-conjugated AGEs (0, 1, 10, 100 µg/mL) was normalized to the group treated with the corresponding concentration of BSA alone ( n = 3 biological replicates). C Immunoblot analysis of phosphorylated-integrin β1 (p-integrin β1) in HMEC-1 cells with Gal-3 (0.33 µM) in the presence or absence of BSA (1.98 µM) or AGEs (1.98 µM). Quantifications were shown below the blots. D Pull-down assays. HMEC-1 cell lysates (100 μg) were pulled down with His-Gal-3 in the presence or absence of BSA or AGEs, the molar ratio of Gal-3 with BSA or AGEs was 1:6. Immunoblot analysis and quantifications of integrin β1 were shown. E Chemical shift changes (Δδ) from these HSQC spectra of Gal-3 and integrin β1. 1 H- 15 N HSQC spectral expansions for 15 N-enriched Gal-3 (20 μM) in the presence of integrin β1 (0.4 μM), plus AGEs (0.4 μM, lower panel). F Bio-Layer interferometry (BLI) analysis of Gal-3-integrin β1 affinity. His-integrin β1 interacted with Gal-3 (200, 400, 600, 800, 1000, 1200, 1400 nM) (left) or Gal-3 (1.4 µM), respectively, plus different concentrations of AGEs (0, 11.2, 22.4, 44.8 µM) (right) at 25 °C. G Representative fluorescence images of condensates formed with GFP-Gal-3 (40 µM) plus BSA or AGEs (240 µM), GFP-Gal-3 (40 µM) + integrin β1 (400 nM) mixture plus BSA or AGEs (240 µM) in PBS (pH 7.4). Quantifications of condensates’ number and diameter in each microscope field were shown ( n = 4 biological replicates). Scale bar, 10 µm. H Particle size of condensates formed by Gal-3 (40 µM) and integrin β1 (400 nM) plus BSA (240 µM) or AGEs (240 µM) in PBS (pH 7.4) ( n = 3 biological replicates). I Confocal microscopy of GFP-Gal-3 (3.3 µM) induced condensates in CHO-K1 cells expressing mCherry-integrin α5 with treatment of BSA (19.8 µM) or AGEs (19.8 µM). The cell indicated by the white arrow was enlarged. Quantifications of the size and the number of the condensates per cell were shown (upper, left to right, n = 5, 6, 6, 5 fields; lower, n = 5 fields; total number of cells were 50–80 in each group). Scale bar, 20 μm. J , K STZ-induced diabetic rats were treated with hydrogels embedded AGEs inhibitor (DM + AGEi) or vehicle (DM + Veh) after wounding. Normal rats treated with blank hydrogels (Normal + Veh) served as the negative control. J Representative images of wounds and percentage of wound closure ( n = 6). K Immunohistochemical staining of CD31(black arrows) in healed wounds. Quantifications of microvessel count per field were shown on the right (left to right, n = 6, 7, 5). Scale bar, 50 µm. All statistical data are presented as means ± SEM. P values were determined by unpaired two-tailed Student’s t test ( A , G , H , J , K ), one-way ANOVA with two-sided Fisher’s LSD post hoc test ( B ) or two-sided Mann–Whitney U test ( I ). Error bars represent the mean ± SEM of each group. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: After blocking with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20), blots were incubated overnight at 4°C with the following primary antibodies: CD31 (1:1000, ab281583, Abcam, USA), β-Actin (1:1000, 612657, BD Biosciences, USA), Gal-3 (1:1000, ab76245, Abcam, USA), collagen I (1:1000, GB114197 , Servicebio, China), collagen III (1:1000, EPR17673 , Abcam, USA), AKT (1:1000, 9272 s, cell signaling technology, USA), p-AKT (Ser473) (1:1000, 9271 s, cell signaling technology, USA), FoxO1 (1:1000, 18592-1-AP, Proteintech, China), integrin β1 (1:1000, 610467, BD Biosciences, USA), integrin α3 (1:1000, 66070, Proteintech, China), integrin α5 (1:1000, sc-10729, Santa Cruz, USA), integrin α6 (1:1000, ab181551, Abcam, USA), p-integrin β1 (Y783) (1:1000, ab62337, Abcam, USA), VEGFR2 (1:1000, 2479S, Cell signaling, USA), p-VEGFR2 (Tyr1175) (1:1000, 2478S, Cell signaling technology, USA), EGFR (1:1000, 4267S, Cell signaling, USA), p-EGFR (Tyr1068) (1:1000, 3777S, Cell signaling technology, USA), integrin αv(1:1000, 27096-1-AP, Proteintech, China), FAK (1:1000, 12636-1-AP, Proteintech China), p-FAK (Y397) (1:1000, 611806, BD Biosciences, USA), CD146 (1:1000, 611208, BD Biosciences, USA), GST (1:1000, KM8005, Sungene Biotech, China), His-tag (1:1000, RM1001, Beijing Ray Antibody Biotech, China), α-Tubulin (1:1000, F0063, Selleck, USA).

Techniques: Recombinant, Concentration Assay, Western Blot, Fluorescence, Microscopy, Confocal Microscopy, Expressing, Negative Control, Immunohistochemical staining, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: In circulation, Gal-3 directly interacts with integrin α5β1 via glycans in vascular endothelial cells, forming a liquid-liquid phase separation, activating downstream FAK, ultimately promoting angiogenesis and skin wound healing. In diabetic states, accumulated AGEs bind to Gal-3, blocking the activation of the integrin α5β1-FAK axis, resulting in reduced angiogenesis and delayed skin wound healing. This figure was created in BioRender. Chen, S. (2025) https://BioRender.com/4tkiilw .

Article Snippet: After blocking with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20), blots were incubated overnight at 4°C with the following primary antibodies: CD31 (1:1000, ab281583, Abcam, USA), β-Actin (1:1000, 612657, BD Biosciences, USA), Gal-3 (1:1000, ab76245, Abcam, USA), collagen I (1:1000, GB114197 , Servicebio, China), collagen III (1:1000, EPR17673 , Abcam, USA), AKT (1:1000, 9272 s, cell signaling technology, USA), p-AKT (Ser473) (1:1000, 9271 s, cell signaling technology, USA), FoxO1 (1:1000, 18592-1-AP, Proteintech, China), integrin β1 (1:1000, 610467, BD Biosciences, USA), integrin α3 (1:1000, 66070, Proteintech, China), integrin α5 (1:1000, sc-10729, Santa Cruz, USA), integrin α6 (1:1000, ab181551, Abcam, USA), p-integrin β1 (Y783) (1:1000, ab62337, Abcam, USA), VEGFR2 (1:1000, 2479S, Cell signaling, USA), p-VEGFR2 (Tyr1175) (1:1000, 2478S, Cell signaling technology, USA), EGFR (1:1000, 4267S, Cell signaling, USA), p-EGFR (Tyr1068) (1:1000, 3777S, Cell signaling technology, USA), integrin αv(1:1000, 27096-1-AP, Proteintech, China), FAK (1:1000, 12636-1-AP, Proteintech China), p-FAK (Y397) (1:1000, 611806, BD Biosciences, USA), CD146 (1:1000, 611208, BD Biosciences, USA), GST (1:1000, KM8005, Sungene Biotech, China), His-tag (1:1000, RM1001, Beijing Ray Antibody Biotech, China), α-Tubulin (1:1000, F0063, Selleck, USA).

Techniques: Blocking Assay, Activation Assay

Journal: Cell communication and signaling : CCS

Article Title: Peritoneal cavity-derived small extracellular vesicles from aged tumor-naïve hosts promote ovarian cancer adhesion and invasion.

doi: 10.1186/s12964-025-02273-1

Figure Lengend Snippet: Fig. 6 Blocking sEV-associated proteins abrogates the enhanced meso-mimetic adhesion observed with aged sEVs. sEVs (5 × 107) purified from perito neal lavage obtained from aged hosts or control (PBS) were incubated with function-blocking antibodies directed against (A) β1 integrin (2 µg), (B) CA125 (MUC16, 1 µg) or (C) LYN kinase (1 µg) in a total volume of 200 ul for 3 h prior to adding to OvCa cells for 24 h. The meso-mimetic adhesion assay was then performed as described in Fig. 3. (D) sEVs (5 × 107) purified from peritoneal lavage obtained from aged hosts or control (PBS) were incubated with the Lyn kinase inhibitor TL0259 (0.1 nM) for 3 h prior to adding to OvCa cells for 24 h. The meso-mimetic adhesion assay was then performed as described in Fig. 3. Assays were performed in triplicate. Data were analyzed using Kruskal-Wallis test and Dunn’s multi-comparison test

Article Snippet: Gels were transferred to a polyvinylidene difluoride membrane (ImmobilonP, Millipore) using a Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell device, and blocked in 5% milk in TBST buffer (25 mmol/l Tris pH 7.5, 150 mmol/l NaCl, 0.1% Tween 20) for 1 h at room temperature (RT), then were incubated overnight with antibodies to CD9, CD63, CD81, TSG101, Annexin A5, Integrin β1, integrin α3, filaggrin, transglutaminase2, Lyn, Mhc1 (Santa Cruz Biotechnology), Integrin α2 (Advanced Cellular Biology), and MUC16/CA125 (Dako) at a 1:100 dilution in 5% milk in TBST at 4 °C with gentle rocking.

Techniques: Blocking Assay, Purification, Control, Incubation, Cell Adhesion Assay, Comparison