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mouse anti integrin β 1 (Santa Cruz Biotechnology)

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Santa Cruz Biotechnology mouse anti integrin β 1
Mouse Anti Integrin β 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 868 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti integrin β 1/product/Santa Cruz Biotechnology
Average 96 stars, based on 868 article reviews

mouse anti integrin β 1 - by Bioz Stars, 2026-06

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( A ) Representative IF and number of CD45 + cells ( n = 4) and mean fluorescence density (AU) for p-S6 ( n = 3–4) and p-Smad2 ( n = 3–4) in 5-week-old WT and mgR mice treated with either IgG2a control or Itgb1 mAb for 1 week starting at 4 weeks of age. Scale bar: 25 μm. ( B ) Schema depicting serologic neutralization experimental design in which mgR mice were treated with Itgb1 mAb for 8 weeks starting at 4 weeks of age with mitral valves analyzed at 12 weeks of age. ( C ) Incidence of MR ( n = 10) and ( D ) morphometric analysis of anterior mitral valve leaflet area ( n = 5). ( E ) Measurement of maximal leaflet thickness ( n = 4–6) and ( F ) mean fluorescence density (AU) for p-S6 ( n = 3) in 12-week-old WT, α5/2, mgR, mgR + Itgb1 MAb, and α5/2 mgR mice. ( G ) Representative Movat pentachrome staining of long-axis sections of mitral valve leaflets and ( H ) representative IF of p-S6 in 12-week-old WT, α5/2, mgR, mgR + Itgb1 MAb, and α5/2 mgR mice. Scale bars: 100 μm. Data are represented as individual values with mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by ( D – F ) 1-way or ( A ) 2-way ANOVA or ( C ) Fisher’s exact test.

Journal: The Journal of Clinical Investigation

Article Title: Integrin-mediated mTOR signaling drives TGF- β overactivity and myxomatous mitral valve degeneration in hypomorphic fibrillin-1 mice

doi: 10.1172/JCI183558

Figure Lengend Snippet: ( A ) Representative IF and number of CD45 + cells ( n = 4) and mean fluorescence density (AU) for p-S6 ( n = 3–4) and p-Smad2 ( n = 3–4) in 5-week-old WT and mgR mice treated with either IgG2a control or Itgb1 mAb for 1 week starting at 4 weeks of age. Scale bar: 25 μm. ( B ) Schema depicting serologic neutralization experimental design in which mgR mice were treated with Itgb1 mAb for 8 weeks starting at 4 weeks of age with mitral valves analyzed at 12 weeks of age. ( C ) Incidence of MR ( n = 10) and ( D ) morphometric analysis of anterior mitral valve leaflet area ( n = 5). ( E ) Measurement of maximal leaflet thickness ( n = 4–6) and ( F ) mean fluorescence density (AU) for p-S6 ( n = 3) in 12-week-old WT, α5/2, mgR, mgR + Itgb1 MAb, and α5/2 mgR mice. ( G ) Representative Movat pentachrome staining of long-axis sections of mitral valve leaflets and ( H ) representative IF of p-S6 in 12-week-old WT, α5/2, mgR, mgR + Itgb1 MAb, and α5/2 mgR mice. Scale bars: 100 μm. Data are represented as individual values with mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by ( D – F ) 1-way or ( A ) 2-way ANOVA or ( C ) Fisher’s exact test.

Article Snippet: Animals were treated with (a) rapamycin (Calbiochem) at 2 mg/kg/d i.p. every other day (q.o.d.) versus DMSO vehicle alone, (b) β 1 integrin (CD29) monoclonal antibody (BioXCell BE0232) at 4 mg/kg/d i.p. q.o.d. versus IgG2a isotype control (BioXCell BE0089), and (c) TGF-β mAb (BioXCell BE0057) at 250 μg i.p. q.o.d. versus IgG1 isotype control (BioXCell #BE0083) for various durations as described, including a final dose 6 hours before euthanasia.

Techniques: Fluorescence, Control, Neutralization, Staining, IF-P

Representative IF and mean fluorescence density (AU) in human normal mitral valves and MVP specimens for ( A ) p-Smad2 and p-S6 ( n = 8), and ( B ) CD45 ( n = 8–9) and β 1 integrin (ITGB1) ( n = 8–10). Scale bars: 50 μm. ( C ) Uniform Manifold Approximation and Projection (UMAP) plots of single-cell RNA-Seq with annotation of primary cell types, demonstrating proportional differences between mitral valves from normal and MVP specimen ( n = 4–6). Volcano plot showcasing DEGs of ( D ) fibroblasts and ( E ) macrophages from normal and MVP valves, with selected DEGs labeled. Genes highlighted in red are upregulated in MVP, while those in blue are upregulated in normal valves. ( F ) Chord diagram illustrating the strength of receptor-ligand signaling interactions among macrophages and fibroblasts in MVP versus normal as reference; *integrins identified as receptors. Data are represented as individual values with mean ± SEM; *** P < 0.001, **** P < 0.0001 by unpaired t test.

Journal: The Journal of Clinical Investigation

Article Title: Integrin-mediated mTOR signaling drives TGF- β overactivity and myxomatous mitral valve degeneration in hypomorphic fibrillin-1 mice

doi: 10.1172/JCI183558

Figure Lengend Snippet: Representative IF and mean fluorescence density (AU) in human normal mitral valves and MVP specimens for ( A ) p-Smad2 and p-S6 ( n = 8), and ( B ) CD45 ( n = 8–9) and β 1 integrin (ITGB1) ( n = 8–10). Scale bars: 50 μm. ( C ) Uniform Manifold Approximation and Projection (UMAP) plots of single-cell RNA-Seq with annotation of primary cell types, demonstrating proportional differences between mitral valves from normal and MVP specimen ( n = 4–6). Volcano plot showcasing DEGs of ( D ) fibroblasts and ( E ) macrophages from normal and MVP valves, with selected DEGs labeled. Genes highlighted in red are upregulated in MVP, while those in blue are upregulated in normal valves. ( F ) Chord diagram illustrating the strength of receptor-ligand signaling interactions among macrophages and fibroblasts in MVP versus normal as reference; *integrins identified as receptors. Data are represented as individual values with mean ± SEM; *** P < 0.001, **** P < 0.0001 by unpaired t test.

Techniques: Fluorescence, RNA Sequencing, Labeling

EVs from GT16 and CV3 were analyzed by MS. The abundance of proteins was compared after normalization based on the total peptide amount according to the human database of Discoverer 2.4. Perseus analysis was performed after focusing on Master Protein, removing contamination, and taking higher than PSMs 4. In the scatter plot, the X-axis represents the log2-transformed normalized abundance of CV3-derived EVs, while the Y-axis represents the log2-transformed normalized abundance of GT16-derived EVs. The plot includes fold change lines at ±2 log2 unit. These lines indicate a 4-fold increase (+2 log2 unit) or a 0.25-fold decrease (−2 log2 unit) in the expression level of GT16-derived EVs relative to CV3-derived EVs. Integrin binding proteins are marked by red circles based on the GO term. Integrin isoforms are marked by blue dots. Other identified proteins are indicated by gray circles.

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: EVs from GT16 and CV3 were analyzed by MS. The abundance of proteins was compared after normalization based on the total peptide amount according to the human database of Discoverer 2.4. Perseus analysis was performed after focusing on Master Protein, removing contamination, and taking higher than PSMs 4. In the scatter plot, the X-axis represents the log2-transformed normalized abundance of CV3-derived EVs, while the Y-axis represents the log2-transformed normalized abundance of GT16-derived EVs. The plot includes fold change lines at ±2 log2 unit. These lines indicate a 4-fold increase (+2 log2 unit) or a 0.25-fold decrease (−2 log2 unit) in the expression level of GT16-derived EVs relative to CV3-derived EVs. Integrin binding proteins are marked by red circles based on the GO term. Integrin isoforms are marked by blue dots. Other identified proteins are indicated by gray circles.

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Transformation Assay, Derivative Assay, Expressing, Binding Assay

The expression of integrins and gangliosides on/in cells and EVs. Cell surface expression of gangliosides, GD3 and GD2, and that of integrins, β 1, β 2, β 3, β 4, β 5, β 6, β 7, β 8, α 2, α 3, α 4, α 5, α 6, and α v , were analyzed by flow cytometry. Anti-GD3 mAb (R24), anti-GD2 mAb (220-51), anti-integrin β 1 ~β 8 Abs, anti-integrin α 2 ~α 6 Abs, or anti-integrin α v Ab were used as primary antibodies, and FITC-labeled secondary antibodies were used ( A ). The expression levels of integrin β 1, α 3, and α v were analyzed in both GD3/GD2(+) and (-) cell-derived EVs by Tim4-bead flow cytometry ( B ). Red lines are results with individual mAbs, and black lines are of negative controls. The expression of integrin β 1 and α 3 was observed in GT16 and CV2 cells and also in EVs derived from them by IB using lysates (1 μg) of both cells and EVs. Anti-integrin β 1 Ab and anti-integrin α 3 Ab were used as primary antibodies, and HRP-labelled secondary Ab was used ( Ci ). The intensity of the obtained bands in Ci was measured and plotted ( Cii,Ciii ). Representative results from repeated experiments (at least 3 times) are presented.

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: The expression of integrins and gangliosides on/in cells and EVs. Cell surface expression of gangliosides, GD3 and GD2, and that of integrins, β 1, β 2, β 3, β 4, β 5, β 6, β 7, β 8, α 2, α 3, α 4, α 5, α 6, and α v , were analyzed by flow cytometry. Anti-GD3 mAb (R24), anti-GD2 mAb (220-51), anti-integrin β 1 ~β 8 Abs, anti-integrin α 2 ~α 6 Abs, or anti-integrin α v Ab were used as primary antibodies, and FITC-labeled secondary antibodies were used ( A ). The expression levels of integrin β 1, α 3, and α v were analyzed in both GD3/GD2(+) and (-) cell-derived EVs by Tim4-bead flow cytometry ( B ). Red lines are results with individual mAbs, and black lines are of negative controls. The expression of integrin β 1 and α 3 was observed in GT16 and CV2 cells and also in EVs derived from them by IB using lysates (1 μg) of both cells and EVs. Anti-integrin β 1 Ab and anti-integrin α 3 Ab were used as primary antibodies, and HRP-labelled secondary Ab was used ( Ci ). The intensity of the obtained bands in Ci was measured and plotted ( Cii,Ciii ). Representative results from repeated experiments (at least 3 times) are presented.

Techniques: Expressing, Flow Cytometry, Labeling, Derivative Assay

Colocalization of GD3 and/or GD2 with integrin β 1 on the cell surface. GD3/GD2(+) GT16 and GD3/GD2(-) CV2 cells (2 × 10 4 ) were seeded on collagen-I-pre-coated glass-bottom dishes and fixed on the bottom of the dish with 4% paraformaldehyde at 70 to 80% confluency. The colocalization of GD3 and integrin β 1 was analyzed by staining with two kinds of primary antibodies, i.e., mouse anti-GD3 mAb (1:100) and rabbit anti-integrin β 1 Ab (1:100), and then by the individual secondary antibodies, Alexa 568-conjugated goat anti-mouse IgG Ab (red) (1:100) and Alexa 488-conjugated goat anti-rabbit IgG (H+L) Ab (1:100) (green), respectively, under a confocal microscope (FLUOVIEW FV10i, Olympus, Tokyo, Japan). ( A ). In the case of the colocalization of GD2 and integrin β 1 on the cell surface, mouse anti-GD2 mAb (1:100) was used with rabbit anti-integrin β 1 Ab (1:100) as primary Abs, and the secondary Abs were as mentioned above ( B ).

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: Colocalization of GD3 and/or GD2 with integrin β 1 on the cell surface. GD3/GD2(+) GT16 and GD3/GD2(-) CV2 cells (2 × 10 4 ) were seeded on collagen-I-pre-coated glass-bottom dishes and fixed on the bottom of the dish with 4% paraformaldehyde at 70 to 80% confluency. The colocalization of GD3 and integrin β 1 was analyzed by staining with two kinds of primary antibodies, i.e., mouse anti-GD3 mAb (1:100) and rabbit anti-integrin β 1 Ab (1:100), and then by the individual secondary antibodies, Alexa 568-conjugated goat anti-mouse IgG Ab (red) (1:100) and Alexa 488-conjugated goat anti-rabbit IgG (H+L) Ab (1:100) (green), respectively, under a confocal microscope (FLUOVIEW FV10i, Olympus, Tokyo, Japan). ( A ). In the case of the colocalization of GD2 and integrin β 1 on the cell surface, mouse anti-GD2 mAb (1:100) was used with rabbit anti-integrin β 1 Ab (1:100) as primary Abs, and the secondary Abs were as mentioned above ( B ).

Techniques: Staining, Microscopy

The close localization of GD3 and/or GD2 with integrin β 1 on EVs as analyzed by double immunostaining. Fresh exosomes (3 μg/200 μL) were added on Tim4-Fc-coated glass-bottom dishes for 30 min at RT. After washing, the bound exosomes were fixed on the bottom of the dish with 4% paraformaldehyde. Fixed exosomes were incubated with primary antibodies (mouse anti-GD3 mAb (1:100) and rabbit anti-integrin β 1 Ab (1:100)) and secondary Abs (Alexa 568-conjugated goat anti-mouse IgG Abs (red) (1:100) and/or Alexa 488-conjugated goat anti-rabbit IgG (H+L) Abs (green) (1:100)) and finally analyzed with a confocal microscope, where the yellow color of the overlapping image indicates the close localization of GD3 and integrin β 1 on GT16-derived EVs ( A upper ) but not on CV2-derived EVs ( A lower ). Colocalization between GD2 and integrin β 1 was examined on EVs using primary antibodies, mouse anti-GD2 mAbs (1:100) and rabbit anti-integrin β 1 Abs (1:100), and the proper secondary Abs as mentioned above. The yellow color of the confocal imaging means close localization between GD2 and integrin β 1 on GT16 cell-derived EVs ( B upper ), but not on CV2-derived EVs ( B lower ).

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: The close localization of GD3 and/or GD2 with integrin β 1 on EVs as analyzed by double immunostaining. Fresh exosomes (3 μg/200 μL) were added on Tim4-Fc-coated glass-bottom dishes for 30 min at RT. After washing, the bound exosomes were fixed on the bottom of the dish with 4% paraformaldehyde. Fixed exosomes were incubated with primary antibodies (mouse anti-GD3 mAb (1:100) and rabbit anti-integrin β 1 Ab (1:100)) and secondary Abs (Alexa 568-conjugated goat anti-mouse IgG Abs (red) (1:100) and/or Alexa 488-conjugated goat anti-rabbit IgG (H+L) Abs (green) (1:100)) and finally analyzed with a confocal microscope, where the yellow color of the overlapping image indicates the close localization of GD3 and integrin β 1 on GT16-derived EVs ( A upper ) but not on CV2-derived EVs ( A lower ). Colocalization between GD2 and integrin β 1 was examined on EVs using primary antibodies, mouse anti-GD2 mAbs (1:100) and rabbit anti-integrin β 1 Abs (1:100), and the proper secondary Abs as mentioned above. The yellow color of the confocal imaging means close localization between GD2 and integrin β 1 on GT16 cell-derived EVs ( B upper ), but not on CV2-derived EVs ( B lower ).

Techniques: Double Immunostaining, Incubation, Microscopy, Derivative Assay, Imaging

Molecular clustering of gangliosides and integrin β 1 . ( A ) The association of GD3 and integrin β 1 and that of GD2 and integrin β 1 on cells was analyzed by IP with anti-integrin β 1 Ab and subsequent IB with anti-GD3 mAb or anti-GD2 mAb as well as anti-integrin β 1 Ab. ( B ) The molecular clustering between GD3 and integrin β 1 and between GD2 and integrin β 1 in EVs derived from them was examined by IP followed by IB as mentioned above.

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: Molecular clustering of gangliosides and integrin β 1 . ( A ) The association of GD3 and integrin β 1 and that of GD2 and integrin β 1 on cells was analyzed by IP with anti-integrin β 1 Ab and subsequent IB with anti-GD3 mAb or anti-GD2 mAb as well as anti-integrin β 1 Ab. ( B ) The molecular clustering between GD3 and integrin β 1 and between GD2 and integrin β 1 in EVs derived from them was examined by IP followed by IB as mentioned above.

Techniques: Derivative Assay

Effects of anti-integrin Abs and anti-GD3/GD2 mAbs on EV function. ( A ) The adhesion activity of GD3/GD2(-) cells to collagen-I examined by RT-CES. Cells (1 × 10 4 ) were cultured in collagen-I-precoated wells containing 100 μL of culture medium and incubated. The data after 24 h ( A ) and 9 h ( B ) of cell incubation are shown. GT16 cell-derived EVs enhanced the adhesion activity of CV2 cells ( A , B ), but anti-integrin β 1 Ab remarkably suppressed the adhesion activity of CV2 cells ( A ). Anti-GD3 mAb and/or anti-GD2 mAb also significantly suppressed the action of EVs to the adhesion of CV2 cells when either one of these anti-ganglioside mAbs was added to CV2 cells together with GD3/GD2(+) cell-derived EVs ( B ). The data until 24 h of incubation ( A ) and until 9 h of incubation ( B ) were analyzed with an unpaired Student’s two-tailed t test. Not significant (NS) p > 0.05, and *** p < 0.001. The mean values ± SD (n = 3) were plotted for each time point.

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: Effects of anti-integrin Abs and anti-GD3/GD2 mAbs on EV function. ( A ) The adhesion activity of GD3/GD2(-) cells to collagen-I examined by RT-CES. Cells (1 × 10 4 ) were cultured in collagen-I-precoated wells containing 100 μL of culture medium and incubated. The data after 24 h ( A ) and 9 h ( B ) of cell incubation are shown. GT16 cell-derived EVs enhanced the adhesion activity of CV2 cells ( A , B ), but anti-integrin β 1 Ab remarkably suppressed the adhesion activity of CV2 cells ( A ). Anti-GD3 mAb and/or anti-GD2 mAb also significantly suppressed the action of EVs to the adhesion of CV2 cells when either one of these anti-ganglioside mAbs was added to CV2 cells together with GD3/GD2(+) cell-derived EVs ( B ). The data until 24 h of incubation ( A ) and until 9 h of incubation ( B ) were analyzed with an unpaired Student’s two-tailed t test. Not significant (NS) p > 0.05, and *** p < 0.001. The mean values ± SD (n = 3) were plotted for each time point.

Techniques: Activity Assay, Cell Culture, Incubation, Derivative Assay, Two Tailed Test

Journal: Virology Journal

Article Title: Replication kinetics of pathogenic Eurasian orthohantaviruses in human mesangial cells

doi: 10.1186/s12985-024-02517-5

Figure Lengend Snippet: Flow cytometry analysis of surface expression of orthohantaviral entry receptors. CIHGM-1 cells were analyzed for the presence of integrin α v β 3 , integrin β 1 , and CD55 on the cell surface. Plots shown are gated on the viable cell population according to scatter profile and the exclusion of Via-Probe™ Cell Viability Solution. Quadrant statistics (Q1-Q4) indicate the percentage of cells in the respective quadrant

Article Snippet: CIHGM-1 cells were washed with PBS, scraped, and stained with phycoerythrin (PE)-conjugated mouse anti-integrin α V β 3 antibody (clone LM609, Millipore) or PE-conjugated mouse anti-integrin β 1 antibody (clone P5D2, R&D Systems, Minneapolis, MN, USA) together with allophycocyanin (APC)-conjugated anti-CD55 (clone IA10, BD Pharmingen, NJ, USA).

Techniques: Flow Cytometry, Expressing

The ectodomain of ICAM-5 and β1 co-immunoprecipitate B35 cells, which had previously been determined to express β1 but not ICAM-5, were treated with recombinant ICAM-5. B35 cells were treated with 1 μg/mL ICAM-5 for 30 min. prior to lysate preparation. Anti-integrin or control (IgG) antibodies (Millipore) were then used to pull out β1 and potential interacting proteins from the ICAM-5 treated B35 cells. Western blot was later performed for ICAM-5 (a) or, as an additional control, β1 (b). ICAM-5 treatment of cells and immunoprecipitation was performed as previously described for MMP-1 (Conant et al. 2004). To determine whether ICAM-5 might interact with β1 in vivo, we also performed studies using lysate from MA treated (6 h) murine hippocampus. As shown in panel c, anti-β1 pulled down both the full length (detected by both N- and C-terminal ICAM-5 antibodies) and ectodomain (detected only by the N-terminal antibody) of ICAM-5. As shown (d), an antibody to the N-terminal domain of ICAM-5 can pull down β1.

Journal: Journal of neurochemistry

Article Title: Methamphetamine-associated cleavage of the synaptic adhesion molecule intercellular adhesion molecule-5

doi: 10.1111/j.1471-4159.2010.07153.x

Figure Lengend Snippet: The ectodomain of ICAM-5 and β1 co-immunoprecipitate B35 cells, which had previously been determined to express β1 but not ICAM-5, were treated with recombinant ICAM-5. B35 cells were treated with 1 μg/mL ICAM-5 for 30 min. prior to lysate preparation. Anti-integrin or control (IgG) antibodies (Millipore) were then used to pull out β1 and potential interacting proteins from the ICAM-5 treated B35 cells. Western blot was later performed for ICAM-5 (a) or, as an additional control, β1 (b). ICAM-5 treatment of cells and immunoprecipitation was performed as previously described for MMP-1 (Conant et al. 2004). To determine whether ICAM-5 might interact with β1 in vivo, we also performed studies using lysate from MA treated (6 h) murine hippocampus. As shown in panel c, anti-β1 pulled down both the full length (detected by both N- and C-terminal ICAM-5 antibodies) and ectodomain (detected only by the N-terminal antibody) of ICAM-5. As shown (d), an antibody to the N-terminal domain of ICAM-5 can pull down β1.

Article Snippet: Anti-β 1 integrin antibodies were obtained from R & D Systems (AF2405) and Millipore Corporation (Bedford, MA, USAAB1952).

Techniques: Recombinant, Control, Western Blot, Immunoprecipitation, In Vivo

The ICAM-5 ectodomain stimulates β1 integrin-dependent phosphorylation of cofilin. B35 cells treated with 1 μg/mL recombinant murine ICAM-5 exodomain (rICAM-5, R & D Systems) for 0–30 min. as indicated (a). As also indicated, select cells were pre-treated with a neutralizing Ab to β1 (Pharmingen, San Diego, CA, USA). It can be appreciated that rICAM-5 stimulated an increase in phospho-cofilin that was inhibited by pre-treatment of cells with the neutralizing antibody. In a separate experiment, primary neurons were treated with 1 μg/mL recombinant murine ICAM-5 as indicated. Lysates from three control and three stimulated culture wells were prepared 60 min later and analyzed by western blot for phospho- and total cofilin (b). Ratios of normalized densitometric data were compared (c), and differences between control and ICAM-5 stimulated were significant (*p < 0.02). In panel d, primary neurons (14 days in vitro) grown on cover slips, were treated for 60 min with medium alone or medium containing 1 μg/mL rICAM-5. Neurons were then fixed and stained using phalloidin or anti-phospho-cofilin as indicated. Although the ICAM-5-associated increase in phospho-cofilin is somewhat generalized, as shown in the merged staining inset, an increase can be appreciated in actin-rich structures having the appearance of dendritic spines (representative spines are indicated by arrows). The scale bar (inset) represents 5 μm.

Journal: Journal of neurochemistry

Article Title: Methamphetamine-associated cleavage of the synaptic adhesion molecule intercellular adhesion molecule-5

doi: 10.1111/j.1471-4159.2010.07153.x

Figure Lengend Snippet: The ICAM-5 ectodomain stimulates β1 integrin-dependent phosphorylation of cofilin. B35 cells treated with 1 μg/mL recombinant murine ICAM-5 exodomain (rICAM-5, R & D Systems) for 0–30 min. as indicated (a). As also indicated, select cells were pre-treated with a neutralizing Ab to β1 (Pharmingen, San Diego, CA, USA). It can be appreciated that rICAM-5 stimulated an increase in phospho-cofilin that was inhibited by pre-treatment of cells with the neutralizing antibody. In a separate experiment, primary neurons were treated with 1 μg/mL recombinant murine ICAM-5 as indicated. Lysates from three control and three stimulated culture wells were prepared 60 min later and analyzed by western blot for phospho- and total cofilin (b). Ratios of normalized densitometric data were compared (c), and differences between control and ICAM-5 stimulated were significant (*p < 0.02). In panel d, primary neurons (14 days in vitro) grown on cover slips, were treated for 60 min with medium alone or medium containing 1 μg/mL rICAM-5. Neurons were then fixed and stained using phalloidin or anti-phospho-cofilin as indicated. Although the ICAM-5-associated increase in phospho-cofilin is somewhat generalized, as shown in the merged staining inset, an increase can be appreciated in actin-rich structures having the appearance of dendritic spines (representative spines are indicated by arrows). The scale bar (inset) represents 5 μm.

Article Snippet: Anti-β 1 integrin antibodies were obtained from R & D Systems (AF2405) and Millipore Corporation (Bedford, MA, USAAB1952).

Techniques: Phospho-proteomics, Recombinant, Control, Western Blot, In Vitro, Staining

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