mouse anti integrin β 1  (Santa Cruz Biotechnology)

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

Journal: Cartilage

Article Title: Semaphorin 7A Regulates the Balance Between Cartilaginous and Fibrous Tissues in the Repair Process of Articular Cartilage Damage

doi: 10.1177/19476035261418126

Figure Lengend Snippet: Integrin β1 (ITGB1) transduces Sema7A signal in chondrocytes. (A) Feature plot showing the expression distribution of PLXNC1 and ITGB1 in the UMAP plot of Figure 1 . (B) P2 Sema7a +/+ and Sema7a -/- chondrocytes were cultured in the presence or absence of neutralizing anti-ITGB1 antibody for 48 hours. Relative mRNA expression of chondrocyte-maker genes was analyzed. Error bars denote mean ± standard error. * P < 0.05, two-tailed Welch’s t test ( n = 6).

Article Snippet: Anti-integrin β1 neutralizing antibody solution (4.3 mg/ml, cat #BE0232, BioXCell, Lebanon, NH) was purchased.

Techniques: Expressing, Cell Culture, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Activation Assay, Membrane, Binding Assay, Incubation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 catalyzes the α2,6-sialylation of LPAR4, EGFR, integrin α5, and integrin β1 in MDA-MB-231 cells. A, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 cells were immunoprecipitated by SSA-agaroses and then blotted with antibodies against LPAR4, EGFR, integrin β1, and integrin α5. The whole-cell lysates were also subjected to WB with indicated antibodies. B, the cell lysates from Con- and ST3GAL4-OE- MDA-MB-231 cells were immunoprecipitated by SSA- and MAM-agaroses and then blotted with antibodies against integrin β1, EGFR, and integrin α5 separately. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the α2,6-sialylated LPAR4, EGFR, integrin α5, or integrin β1 versus total LPAR4, EGFR, integrin α5, or integrin β1, respectively) in ( A ) and (the α2,3-sialylated or α2,6-sialylated integrin β1, EGFR, or integrin α5 versus total integrin β1, EGFR, or integrin α5, respectively) in ( B ) are shown as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test and two-tail unpaired t test, respectively). WB, Western blot; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; SSA, Sambucus sieboldiana agglutinin; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; Maackia amurensis.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Immunoprecipitation, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 mediates integrin β1–LPAR4/EGFR complex formation. A and B, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 ( A ) and BT549 ( B ) cells were immunoprecipitated by anti-integrin β1 antibody and then blotted with antibodies against LPAR4, EGFR, and integrin β1. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the association of EGFR or LPAR4 with integrin β1, respectively) in ( A ) and ( B ) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). ST6GAL1, β-galactoside α2,6-sialyltransferase 1; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; WB, Western blot.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Immunoprecipitation, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Schematic diagram of the proposed molecular mechanism for negative regulation of Hippo signaling via ST6GAL1. Various upstream cell membrane receptors of the Hippo pathway have been identified, including the RTKs ( e.g. , EGFR), GPCRs ( e.g. , LPAR4), and integrins ( e.g. , integrin α5β1). The RTK, GPCR, and integrin signals transduced by growth factors (GFs, e.g. , EGF), extracellular factors ( e.g. , LPA), and the extracellular matrix (ECM, e.g. , FN) can facilitate Hippo pathway effectors ( e.g. , PI3K and FAK) association, which promote LATS1/2-mediated regulation of YAP. In the cells with ST6GAL1 expression ( left ), the cell membrane receptors, such as EGFR, LPAR4, and integrin α5β1, are modified by α2,6-sialylation, which mediate the integrin β1–EGFR/LPAR4 complex formation and in turn facilitate their responses to EGF, LPA, and FN, respectively. These signalings inactivate LATS1/2 kinases or induce the dephosphorylation of YAP, finally leading to hypophosphorylated YAP (p-YAP S127). Hypophosphorylated YAP accumulates in the nucleus, where it can bind to various transcription factors (TFs, e.g. , TEAD family) to enhance the expression of target genes ( e.g. , ANKRD1 , CTGF , and CYR61 ) expression that promote cell adhesion, spreading, proliferation, migration, and metastasis. The Hippo signaling can be inhibited by the verteporfin (VP) inhibitor, which targets YAP-TEAD activity. In the ST6GAL1 deficiency cells ( right ), the N -glycans on cell membrane receptors are without α2,6-sialylation, which exhibit weak integrin β1–EGFR/LPAR4 complex formation and delayed responses to EGF, LPA, and FN stimulation and activate the LATS1/2 kinases and phosphorylate YAP on S127. The phosphorylated YAP (p-YAP S127) is retained in the cytoplasm, inhibiting YAP/TEAD-dependent transcription. The p of the red background represents the activation of related proteins, while gray background represents the inactivation. LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FN, fibronectin; GPCR, G protein–coupled receptor; GT, glycosyltransferase; LPA, lysophosphatidic acid; FAK, focal adhesion kinase.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Membrane, Expressing, Modification, De-Phosphorylation Assay, Migration, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were incubated in isotonic media, in hypotonic media for 5, 15, 30 and 60 minutes or under Recovery conditions in which hypotonic media was replaced after 60 minutes with isotonic media for 5, 15, 30 or 60 minutes. Cells were labeled for Cav1, fixed and imaged by TIRF widefield imaging. Average Cav1 intensity per cell was quantified and normalized to isotonic control. Representative images of select conditions are shown. (n=3 independent experiments with >30 cells for each condition; ANOVA with Dunnett’s post-test comparing to isotonic control; **p < 0.01; ***p < 0.001; Scale bar: 2µm). B) STED imaging of anti-Cav1 labeling MDA-MB-231 cells incubated in isotonic media or in hypotonic media for 15 or 60 minutes. Cav1 endocytic vacuoles were counted per cell. (n>28 from three independent experiments for isotonic and hypotonic 15 minutes, four independent experiments for other conditions; (ANOVA with Dunnett post-test comparing to isotonic control; *p < 0.05; ****p < 0.0001; Scale bar: 4µm; Inset scale bar: 1µm). C) MDA-MB-231 cells incubated with isotonic or hypotonic media for 60 minutes were fixed and labeled for Cav1, CD44 and β1-integrin (Scale bar: 4µm). The diameter of individual Cav1, CD44, and β1-integrin positive endosomes was measured from three independent experiments.

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Incubation, Labeling, Imaging, Control

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were incubated with anti-CD44 and anti-β1 integrin antibodies in isotonic media for 60 minutes at 4°C or 37°C or in hypotonic media for 5, 15, 30 or 60 minutes at 37°C. As indicated, an acid wash treatment was performed at 4°C to remove surface antibodies and cells were fixed and labeled with secondary antibodies to anti-CD44 and anti-β1 integrin as well as for endogenous Cav1. (n= >30 cells per condition from three independent experiments; ANOVA with Dunnett post-test comparing each condition to isotonic 4°C control; *p < 0.05; ***p < 0.001; ****p < 0.0001; Scale bars: 2 µm). B) MDA-MB-231 cells were transfected with wild-type CDC42-GFP or dominant negative (Dom Neg) CDC42-GFP, incubated with isotonic or hypotonic media for 60 minutes and then fixed and labeled for Cav1 and CD44. CDC42-GFP was also imaged and the number of Cav1 endocytic vacuoles counted per cell (n>30 cells from three independent experiments; ANOVA with Dunnett’s post-test comparing to isotonic control; ****p < 0.0001; Scale bar: 2µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Incubation, Labeling, Control, Transfection, Dominant Negative Mutation