mouse mab against yap  (Proteintech)

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: α2,3-sialylation regulates the Hippo pathway through competing with α2,6-sialylation. A, the cell lysates from ST3GAL4-3xFLAG-overexpressing (ST3GAL4-OE) MDA-MB-231 cells were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, anti-FLAG, anti-ST6GAL1, and anti-GAPDH antibodies. B, the cell membrane fractions from Con and ST3GAL4-OE cells were blotted with ConA, RCA-I (recognizing terminal galactose), MAA, and SNA lectins. C, cell lysates from the MDA-MB-231 derivative cell lines as indicated (The ST6GAL1 KO + ST3GAL4 OE stable cell line was established by overexpressing ST3GAL4 in ST6GAL1 KO cells.) were immunoblotted with indicated antibodies as mentioned in ( A ). The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1) in ( A ) and ( C ) are shown as the mean ± SD ( n = 3 biological replicates, n.s. not statistically significant, p > 0.05, ∗∗∗, p < 0.001, ∗∗∗∗, p < 0.0001 are determined by two-tail unpaired t test and one-way ANOVA with Tukey's post hoc test, respectively). D, the cell membrane fractions from the cells mentioned in ( C ) were blotted with ConA, RCA-I, MAA, and SNA lectins. LATS, large tumor suppressor kinase; SNA, Sambucus nigra ; YAP, yes-associated protein; RCA-I, Ricinus communis agglutinin I; ConA, Concanavalin A; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; MAA, Maackia amurensis agglutinin; Con, control.

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, Stable Transfection, 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: 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: Scientific Reports

Article Title: Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus

doi: 10.1038/s41598-022-13777-y

Figure Lengend Snippet: Establishment and characterization of Bactrian camel and llama AEC cultures. ( A ) Immunofluorescence analysis showing the development of tight-junctions (ZO-1, white) and ciliogenesis (β-tubulin, red) in Bactrian camel and llama AEC cultures over time from 1-day to 4 weeks post ALI exposure. The cells were counterstained with DAPI (blue) to visualize the nuclei. ( B,C ) Ciliogenesis quantification of camel and llama AEC cultures overtime, respectively. Ciliation was quantified by measuring the area above a fluorescence intensity threshold of five random images acquired per condition. ( D ) Transepithelial electrical resistance (TEER) measurement of camel and llama AEC cultures overtime during the differentiation. ( E ) Epithelial morphology of ex vivo tissues (upper panel) and well-differentiated camel and llama AEC cultures (lower panel). ( F ) DPP4 expression in well-differentiated camel and llama AEC cultures, with Vero cells as a positive control. Scale bar is 20 µm.

Article Snippet: Alexa-Fluor ® 647-labelled rabbit anti β-tubulin IV (Cell Signalling Technology, 9F3) and Alexa-Fluor ® 594-labelled mouse antibody against ZO-1 (Thermo Fisher Scientific, 1A12) were used to visualize cilia and tight junctions, respectively.

Techniques: Immunofluorescence, Fluorescence, Ex Vivo, Expressing, Positive Control

Journal: Scientific Reports

Article Title: Brick Strex: a robust device built of LEGO bricks for mechanical manipulation of cells

doi: 10.1038/s41598-021-97900-5

Figure Lengend Snippet: Manual cell stretching and compression experiment with Brick Strex S. Epithelial cell monolayers were grown on silicon membranes and subjected to stretching or compression in Brick Strex S device. ( a ) Epithelial stretching experiment, where epithelium was subjected to a 25% strain. ( b ) Representative maximum intensity Z-projection confocal microscopy images showing cells before (relaxed substrate) and after stretching (2 h after stretching). Cell were stained with DAPI to highlight the nuclei (magenta) and immunolabelled against \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin (green). ( c ) Quantification of the nucleo-cytoplasmic ratio of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin after single stretch of the substrate in comparison to unstretched control samples. ( d ) Lateral compression experiment of the epithelial monolayer. ( e ) Cells were stained with DAPI to highlight the nuclei (magenta) and immunolabelled against YAP1 (green). ( f ) Quantification of the nucleo-cytoplasmic ratio of YAP1 in response to compression. ( g ) Similar lateral compression experiments as shown in e. The cells were stained with DAPI to highlight the nuclei (magenta) and immunolabeled against \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin (green). ( h ) blow-up images of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin staining shown in g. Scale bars 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document} μ m. Error bars represent the standard error of the mean (SEM). Statistical analyses were performed using an un-paired Student´s t-test when comparing between cells before and after stretching/compression (****p < 0.0001; **p < 0.05).

Article Snippet: Immunostaining of the samples was done within the attached cell culture columns by using a primary rabbit antibody against \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin (1:2000 in 3% BSA-PBS, ab6302, Abcam, Cambridge, UK), a mouse monoclonal antibody against YAP1 (1:500 in 3% BSA-PBS, YAP163.7, sc-101199, Santa Cruz Biotechnology, Dallas, USA) and Alexa 568-conjugated phalloidin to detect actin (1:100 in 3% BSA-PBS, 1 h in RT, in dark) followed by Alexa 488-conjugated goat anti-mouse and Alexa 647-conjugated goat anti-rabbit secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Confocal Microscopy, Staining, Comparison, Control, Immunolabeling

Journal: Scientific Reports

Article Title: Brick Strex: a robust device built of LEGO bricks for mechanical manipulation of cells

doi: 10.1038/s41598-021-97900-5

Figure Lengend Snippet: Western blot analysis of lateral compression -induced effects on YAP1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin. Epithelial cells were cultured on a pre-strained PDMS membrane in Brick Strex S device until full confluency. Following sequential membrane relaxation and increased lateral compression, the cells were let to recover for 2 h prior to cell lysis and analyzed by western blot (n = 2). ( a ) Expression and phosphorylation (S127P) status of YAP1 and ( b ) expression of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin were analyzed from whole cell lysates of non-compressed (before) and compressed (after) samples (20 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document} μ g of total protein per sample). Arrows indicate the compression-induced cleavage products of YAP1. Asterisk indicates the presence of full-length (FL) and higher molecular weight \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin in the compressed sample. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -actin was used as an internal control. See also full-length blots in Supplementary Fig. 8.

Article Snippet: Immunostaining of the samples was done within the attached cell culture columns by using a primary rabbit antibody against \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upbeta$$\end{document} β -catenin (1:2000 in 3% BSA-PBS, ab6302, Abcam, Cambridge, UK), a mouse monoclonal antibody against YAP1 (1:500 in 3% BSA-PBS, YAP163.7, sc-101199, Santa Cruz Biotechnology, Dallas, USA) and Alexa 568-conjugated phalloidin to detect actin (1:100 in 3% BSA-PBS, 1 h in RT, in dark) followed by Alexa 488-conjugated goat anti-mouse and Alexa 647-conjugated goat anti-rabbit secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Western Blot, Cell Culture, Membrane, Lysis, Expressing, Phospho-proteomics, Molecular Weight, Control