integrin g2 af1740 r d systems  (R&D Systems)

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: Cell reports

Article Title: Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis

doi: 10.1016/j.celrep.2023.113240

Figure Lengend Snippet: (A) WT or CD11b −/− mice were challenged i.v. with 25,000 viable cells of WT or ece1 Δ/Δ C. albicans , and cerebral clearance was assessed over 20 days. (B–D) Schematic diagram of fungistasis assay (B) and aggregate data of candidalysin-activated BV-2 cells challenged with either WT or ece1 Δ/Δ C. albicans with and without anti-CD11b or NIF blockade of CD11b (C and D). (E and F) Fungistasis assay of candidalysin-activated (E) primary microglia or (F) primary astrocytes from WT mice with WT C. albicans with and without anti-CD11b or NIF blockade of CD11b. (G) Pull-down assays using CD11-expressing CHO cells. CHO-CD11a/b/c cell lysates (prey) were incubated with biotinylated candidalysin (Bio-Clys; bait) or biotinylated serine (Bio-serine; control), and the mixtures were loaded onto prewashed streptavidin beads. Bait-prey complexes were eluted from the beads and loaded onto SDS-PAGE gels to detect CD11a, CD11b, and CD11c via western blotting. (H–J) Schematic diagrams and aggregate data depicting in vitro assays in which the dose-dependent binding of plate-bound (H) CD11b extracellular domain or (I) candidalysin/SC peptide to the other reagent or (J) CD11b or CD11c extracellular domains binding to plate-bound candidalysin were determined colorimetrically (OD, optical density). (K) Flow cytometric analysis of splenocytes from WT or CD11b −/− mice incubated with AF647-conjugated CL (10 μM). n = 4, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way ANOVA followed by Tukey’s test for multiple comparison (A–F), two tailed Student’s t test (H–J), or two-way ANOVA (K). Data are representative of two or three independent experiments.

Article Snippet: Plates were blocked with i-Block (2%) for 2 h at 37°C and incubated with a 2/3 serial dilution of Histagged CD11b (7959-AM, R&D systems, Minneapolis, MN) or CD11c (7987-AX, R&D systems, Minneapolis, MN), starting at 50nM for 2 h at 37°C.

Techniques: Expressing, Incubation, Control, SDS Page, Western Blot, In Vitro, Binding Assay, Comparison, Two Tailed Test

Journal: Cell reports

Article Title: Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis

doi: 10.1016/j.celrep.2023.113240

Figure Lengend Snippet:

Article Snippet: Plates were blocked with i-Block (2%) for 2 h at 37°C and incubated with a 2/3 serial dilution of Histagged CD11b (7959-AM, R&D systems, Minneapolis, MN) or CD11c (7987-AX, R&D systems, Minneapolis, MN), starting at 50nM for 2 h at 37°C.

Techniques: Recombinant, Control, Lysis, Magnetic Beads, Software, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis

doi: 10.1016/j.celrep.2023.113240

Figure Lengend Snippet: (A) WT or CD11b −/− mice were challenged i.v. with 25,000 viable cells of WT or ece1 Δ/Δ C. albicans , and cerebral clearance was assessed over 20 days. (B–D) Schematic diagram of fungistasis assay (B) and aggregate data of candidalysin-activated BV-2 cells challenged with either WT or ece1 Δ/Δ C. albicans with and without anti-CD11b or NIF blockade of CD11b (C and D). (E and F) Fungistasis assay of candidalysin-activated (E) primary microglia or (F) primary astrocytes from WT mice with WT C. albicans with and without anti-CD11b or NIF blockade of CD11b. (G) Pull-down assays using CD11-expressing CHO cells. CHO-CD11a/b/c cell lysates (prey) were incubated with biotinylated candidalysin (Bio-Clys; bait) or biotinylated serine (Bio-serine; control), and the mixtures were loaded onto prewashed streptavidin beads. Bait-prey complexes were eluted from the beads and loaded onto SDS-PAGE gels to detect CD11a, CD11b, and CD11c via western blotting. (H–J) Schematic diagrams and aggregate data depicting in vitro assays in which the dose-dependent binding of plate-bound (H) CD11b extracellular domain or (I) candidalysin/SC peptide to the other reagent or (J) CD11b or CD11c extracellular domains binding to plate-bound candidalysin were determined colorimetrically (OD, optical density). (K) Flow cytometric analysis of splenocytes from WT or CD11b −/− mice incubated with AF647-conjugated CL (10 μM). n = 4, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way ANOVA followed by Tukey’s test for multiple comparison (A–F), two tailed Student’s t test (H–J), or two-way ANOVA (K). Data are representative of two or three independent experiments.

Article Snippet: Plates were blocked with i-Block (2%) for 2 h at 37°C and incubated with a 2/3 serial dilution of Histagged CD11b (7959-AM, R&D systems, Minneapolis, MN) or CD11c (7987-AX, R&D systems, Minneapolis, MN), starting at 50nM for 2 h at 37°C.

Techniques: Expressing, Incubation, Control, SDS Page, Western Blot, In Vitro, Binding Assay, Comparison, Two Tailed Test

Journal: Cell reports

Article Title: Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis

doi: 10.1016/j.celrep.2023.113240

Figure Lengend Snippet:

Article Snippet: Plates were blocked with i-Block (2%) for 2 h at 37°C and incubated with a 2/3 serial dilution of Histagged CD11b (7959-AM, R&D systems, Minneapolis, MN) or CD11c (7987-AX, R&D systems, Minneapolis, MN), starting at 50nM for 2 h at 37°C.

Techniques: Recombinant, Control, Lysis, Magnetic Beads, Software, Enzyme-linked Immunosorbent Assay