Journal: Frontiers in Oncology

Article Title: Inter and intra-tumor heterogeneity of paediatric type diffuse high-grade gliomas revealed by single-cell mass cytometry

doi: 10.3389/fonc.2022.1016343

Figure Lengend Snippet: Summary of the 15 antibodies used for the mass cytometry analysis.

Article Snippet: Anti-Human CD49c , 161Dy , Fluidigm , 3161016B , .

Techniques: Mass Cytometry

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrin expression in clone C cells. (A) Expression patterns of integrin β1, αv, α6, and α1 in confluent monolayers of clone C cells seeded at LD and HD. Integrin staining is depicted in red, and nuclear staining with Sytox is shown in green. Integrin α3 staining was also observed by immunostaining but not depicted here. (B) Integrin expression in clone C cells was investigated by basolateral biotinylation followed by immunoprecipitation with integrin antibodies as indicated and Western blotted with anti-streptavidin antibodies (for αv and α3 in the top panel and all lanes in the bottom panel). In addition, Western blots were performed on biotin-streptavidin–purified basolateral membranes from HD cells with integrin α1, α5, and α6. (C) Western blots of cell lysates from LD and HD cells using the polyclonal antibody directed against extracellular domain of rabbit integrin β1 (left). (Right) Western blot using the same antibody in the presence of 600 ng/ml recombinant peptide immunogen. (D) Flow cytometry analysis of clone C cells with various integrin antibodies as indicated in the labels.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Expressing, Staining, Immunostaining, Immunoprecipitation, Western Blot, Purification, Recombinant, Flow Cytometry

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrin β1 binds to hensin and integrin αV and gets tyrosine phosphorylated in high density cells. (A) In the first panel, 140 μg of DTSSP–cross-linked cell protein from LD and HD phenotypes were immunoprecipitated with anti–integrin β1 CSAT antibodies, and the blots were probed with guinea pig anti-hensin antibody. In control experiment, membrane containing integrin β1 immunoprecipitates described in the first panel was stripped and reprobed with a different anti–integrin β1 antibody MAB1965 (second panel). Chemically cross-linked cell lysates from LD and HD cells were immunoprecipitated with goat anti-hensin/DMBT1 antibody and blotted with anti–integrin β1 MAB1965 antibody (third panel). Control experiment in which the same samples were probed with guinea pig anti-hensin antibody (fourth panel). (B) Tyrosine phosphorylation of integrin β1. (Left) Anti-phosphotyrosine Western blot of integrin β1 immunoprecipitates (described in A) from LD and HD. (Middle) Control Western blot with integrin β1 antibody. (Right) A streptavidin Western blot of biotinylated basolateral membrane proteins (from HD) that were immunoprecipitated with anti–integrin β1 antibody. (C) Interaction of integrin β1 with integrin α subunits was examined by Western blotting of integrin β1 immunoprecipitates with antibodies to integrin αv, α6, and α1 (left lane in first three panels) and α3 and α5 (last two lanes). Interaction of hensin with integrin α subunits was examined by Western blotting of hensin immunoprecipitates with antibodies to integrin αv, α6, and α1 (right lane in first three panels) from HD cells. (D) Interaction of integrin αv with integrin β subunits was examined by Western blotting of integrin αv immunoprecipitates with integrin β1 (first lane), integrin β3 (second lane), and integrin β5 (third lane) antibodies.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Effect of anti-integrin antibodies on cell height and cross-sectional area a

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques:

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Role of integrin αv and β1 in terminal differentiation of clone C epithelia. (A) Clone C cells cultured at high density in media containing integrin αv and β1 function-blocking antibodies or integrin α1 function-blocking antibody. Apical and basal actin stained with phalloidin (red) and imaged by confocal microscopy showing en face and XZ optical sections. (B) HD cells cultured with or without integrin function-blocking antibodies as indicated in the labels were examined for their ability to endocytose fluorescein-dextran. Fluorescein-dextran in green and nuclear stain (Sytox red) in red. (C, top) Clone C cells seeded at low density and allowed to form confluent monolayers, when examined by F-Actin staining, have stress fibers on the basal surface and very little apical actin staining (left); however, when the same experiment is carried out in the presence of integrin β1–activating antibody P4G11, apical actin staining was observed with a marked difference in stress fiber staining on the basal surface. (Bottom) LD cells cultured in the absence or presence of integrin β1–activating antibody P4G11 were examined for their ability to endocytose green fluorescence (FITC) dextran. Nucleus was visualized with Sytox Red dye.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Cell Culture, Blocking Assay, Staining, Confocal Microscopy, Fluorescence

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrins αv and β1 mediate ECM deposition of hensin. (A) ECM hensin staining: Clone C cells seeded at high density were cultured with various function-blocking integrin antibodies and exposed to anti-hensin antibodies (followed by rhodamine secondary antibodies) before fixation or permeabilization. Nuclear staining is shown in green. (B) Control experiment to examine intracellular hensin: Clone C cells seeded at high density were cultured with function-blocking integrin antibodies and exposed to anti-hensin antibodies after fixation and permeabilization. (C) Hensin secretion in basolateral medium: Equal amounts of combined basolateral medium from HD cells cultured in the presence and absence of integrin αv–and β1–blocking antibodies collected for 3 d was precleared with agarose beads, immunoprecipitated with anti-hensin (DMBT1) antibodies, and probed with anti-hensin (SRCR6/7) antibodies. (D) Clone C cells seeded at low density and cultured in the absence or presence of activating antibodies to integrin β1 and stained with hensin antibodies before (top) and after (bottom) permeabilization. (E) ECM was extracted from clone C cells seeded at identical low densities but cultured in the absence or presence of integrin β1–activating antibodies for 5 d. The ECM samples were then probed with Western blotting with guinea pig anti-hensin antibody.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Staining, Cell Culture, Blocking Assay, Immunoprecipitation, Western Blot

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrin α6 is a candidate for transmitting signals from ECM hensin to the cell interior. Effect of function-blocking α6 integrin antibodies on clone c cells seeded at low density on preformed hensin matrix and cultured for 5 d. Monolayers were then stained with Phalloidin (red) and Sytox (green), and the combined projection of all confocal sections is presented in these panels. XZ section represents a typical cross-section through the monolayer.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Blocking Assay, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Mouse-Induced Pluripotent Stem Cells Differentiate into Odontoblast-Like Cells with Induction of Altered Adhesive and Migratory Phenotype of Integrin

doi: 10.1371/journal.pone.0080026

Figure Lengend Snippet: Cells were cultured in the absence (control: grey bars) or presence (black bars) of CS (10% Col-I) and BMP-4 for 7 days in the presence of various anti-integrin monoclonal antibodies (as shown). Detection of the relevant secondary antibody was performed by flow cytometry to estimate the numbers of cells expressing each protein. Negative control values (secondary antibody alone) were subtracted from test values to give the mean fluorescence intensity. Data are mean ± SD (n=3).

Article Snippet: These mAbs were anti-mouse integrin β (Ha2/11), anti-mouse integrin α (Ha31/8), anti-mouse integrin α2 (Ha1/29), anti-mouse integrin α3 (clone 42; BD Biosciences, San Jose, CA USA), anti-mouse integrin α5 (6F4), anti-rat integrin α6 (GoH3; Santa Cruz Biotechnology Inc.), anti-mouse integrin α7 (Cy8), anti-mouse integrin αV (L230), and anti-mouse integrin αVβ3 (23C6; Santa Cruz Biotechnology Inc.).

Techniques: Cell Culture, Flow Cytometry, Expressing, Negative Control, Fluorescence

Journal: PLoS ONE

Article Title: Mouse-Induced Pluripotent Stem Cells Differentiate into Odontoblast-Like Cells with Induction of Altered Adhesive and Migratory Phenotype of Integrin

doi: 10.1371/journal.pone.0080026

Figure Lengend Snippet: (A) The expression of odontoblastic marker mRNAs (DSPP and Dmp-1) in CS/BMP-4 differentiated iPS cells and in E14Tg2a odontoblastic cells was assessed by RT-PCR following culture in the presence of various anti-integrin monoclonal antibodies (5 μg/ml). No significant cross-reactivity with other integrin proteins was observed for these antibodies. Images shown are representative of at least three independent experiments. (B) The effect of transfection of iPS cells and E14Tg2a cells with an integrin α2-specific siRNA. Images show RT-PCR analysis of integrin α2 mRNA expression in cells 24 h after transfection with siRNA (top panels), with expression of the housekeeping gene, GAPDH, as a control (second panels). Western blot analysis (lower three panels) show integrin α2 protein expression in these cells 24 h after siRNA transfection. (C) As in B, iPS cells and E14Tg2a cells were treated with an integrin α2-specific siRNA and the expression of DSPP and Dmp-1 mRNA and protein were measured.

Article Snippet: These mAbs were anti-mouse integrin β (Ha2/11), anti-mouse integrin α (Ha31/8), anti-mouse integrin α2 (Ha1/29), anti-mouse integrin α3 (clone 42; BD Biosciences, San Jose, CA USA), anti-mouse integrin α5 (6F4), anti-rat integrin α6 (GoH3; Santa Cruz Biotechnology Inc.), anti-mouse integrin α7 (Cy8), anti-mouse integrin αV (L230), and anti-mouse integrin αVβ3 (23C6; Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot

Journal: PLoS ONE

Article Title: Mouse-Induced Pluripotent Stem Cells Differentiate into Odontoblast-Like Cells with Induction of Altered Adhesive and Migratory Phenotype of Integrin

doi: 10.1371/journal.pone.0080026

Figure Lengend Snippet: Effect on ALP activity. CS/BMP-4-differentiated iPS cells and E14Tg2a cells were treated with either integrin α2-specific siRNA (or control siRNA) or anti-integrin α2 mAbs (or control mAbs). ALP activity data are presented as the mean ± SD (n=4) of the absorbance at 405 nm, normalized against total protein.

Article Snippet: These mAbs were anti-mouse integrin β (Ha2/11), anti-mouse integrin α (Ha31/8), anti-mouse integrin α2 (Ha1/29), anti-mouse integrin α3 (clone 42; BD Biosciences, San Jose, CA USA), anti-mouse integrin α5 (6F4), anti-rat integrin α6 (GoH3; Santa Cruz Biotechnology Inc.), anti-mouse integrin α7 (Cy8), anti-mouse integrin αV (L230), and anti-mouse integrin αVβ3 (23C6; Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Mouse-Induced Pluripotent Stem Cells Differentiate into Odontoblast-Like Cells with Induction of Altered Adhesive and Migratory Phenotype of Integrin

doi: 10.1371/journal.pone.0080026

Figure Lengend Snippet: (A, B) The adhesion of differentiated (black bars) and undifferentiated (white bars) iPS cells to a substratum of either Fn (5 μg/ml; A) or Col-I (1 μg/ml; B) was assayed in the presence of the indicated anti-integrin antibodies. Data are the number of adherent cells, expressed as a percentage of the total number of cells. Bars indicate the standard deviation. (C, D) Similar experiments were used to investigate motility. The migration of differentiated (black bars) or undifferentiated cells (grey bars) through Transwell inserts coated with Fn (5 μg/ml; C) or Col-I (1 μg/ml; D) was assayed in the presence of the indicated anti-integrin antibodies. Cells were added to the upper chamber and incubated for 3 h. Motility was estimated by counting the number of cells that had migrated to the undersides of the membranes. Data presented are the mean ± SD of at least 10 random microscopic fields.

Article Snippet: These mAbs were anti-mouse integrin β (Ha2/11), anti-mouse integrin α (Ha31/8), anti-mouse integrin α2 (Ha1/29), anti-mouse integrin α3 (clone 42; BD Biosciences, San Jose, CA USA), anti-mouse integrin α5 (6F4), anti-rat integrin α6 (GoH3; Santa Cruz Biotechnology Inc.), anti-mouse integrin α7 (Cy8), anti-mouse integrin αV (L230), and anti-mouse integrin αVβ3 (23C6; Santa Cruz Biotechnology Inc.).

Techniques: Standard Deviation, Migration, Incubation