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snaka51 (R&D Systems)

Name: snaka51
Brand: R&D Systems
Rating: 92 (6 reviews)

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R&D Systems snaka51
Snaka51, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/snaka51/product/R&D Systems
Average 92 stars, based on 6 article reviews

snaka51 - by Bioz Stars, 2026-02

92/100 stars

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Journal: bioRxiv

Article Title: Fibrillar adhesion dynamics govern the timescales of nuclear mechano-response via the vimentin cytoskeleton

doi: 10.1101/2023.11.08.566191

Figure Lengend Snippet: a. Example images of YAP stained cells after 30min of indicated pharmacological treatment; scale bar 25μm. b. Quantification of N/C YAP ratio after 30min pharmacological treatment. (n=121/42/60/48/57 cells for DMSO/bleb/cytoD/Y-27/latA from at least 3 independent experiments; Kruskal-wallis analysis of variance (ANOVA) with Dunn’s multiple comparison test). c. Example colour maps of traction forces after 30min pharmacological treatment; scale bar 50μm. d. Quantification of cell tractions on 15kPa polyacrylamide gels. (n=16/22/21/26/17 cells for DMSO/bleb/cytoD/Y-27/latA from 2 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). e. Examples of integrin-α 5 snaka51 clone stained cells after 30min of the indicated pharmacological treatment; scale bar 25μm / zoom 5μm. f. Quantification of the percentage area under the nucleus occupied by fibrillar adhesions. (n=52/46/48/45/76 cells for DMSO/bleb/cytoD/Y-27/latA from at least 3 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). g. Correlation between N/C YAP ratio and percentage area of fibrillar adhesions under the nucleus for different drug treatments (colour coded) with different incubation times (size coded). (At least 3 independent experiments with a minimum of 28 cells per condition). h. Example images of mechano-reporter L_NLS-41-GFP transfected cells prior to and during cytoD or latA treatment; scale bar 25μm. i. Quantification of change in N/C L_NLS-41-GFP ratio with time (normalized to the pre-treatment ratio) for cells upon with indicated pharmacological treatment. (n=40/33/30 cells for DMSO/cytoD/latA from 3 independent experiments). j. Examples of LaminB stained nuclei after 1 hour of the indicated drug treatment. The yellow line signifies the position of the Z-plane re-slice; scale bar 5µm. k. Quantification of the nuclear height after 1hr pharmacological treatment. (n=69/70/70 nuclei for DMSO/cytoD/latA from 5 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test)

Article Snippet: Integrin α5, clone Snaka51 (1:300, MABT201, Millipore).

Techniques: Staining, Comparison, Incubation, Transfection

a. Example images of integrin-α 5 , clone snaka51 stained cells in scramble or PUR4 peptide seeded for 4 hours in presence of 500nM peptide. Scale bar 20µm, zoom 5µm. b. Percentage area under the nucleus occupied by fibrillar adhesions for scramble and PUR4 peptide. (n=74/109 cells for scramble/PUR4 from 4 independent experiments; unpaired t-test). c. Example images of YAP stained cells treated for 30min with indicated pharmacological treatment for scramble and PUR4 peptide. Scale bar 20µm. d. Quantification of N/C YAP ratio for cells in scramble or PUR4 peptide conditions treated with 30min pharmacological treatment. (Scramble – n=80/74/82 cells for DMSO/bleb/cytoD. PUR4 – n=93/118/110 cells for DMSO/bleb/cytoD from at least 3 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). e. Example images of mechano-reporter L_NLS-41-GFP transfected cells in scramble or PUR4 peptide at different time points are treatment with cytoD. Scale bar 20µm. f. Quantification of change in N/C L_NLS-41-GFP ratio (normalized to the pre-cytoD treatment ratio) for scramble (grey) and PUR4 (blue) peptide conditions treated with cytoD. Scramble n=36, PUR4 n=44 cells from 3 independent experiments. g. Example images of LaminB stained nuclei for scramble and PUR4 cells treated with DMSO control or CytoD. Yellow line indicates the position of the re-slice; sale bar 5µm. h. Quantification of nuclear height for scramble and PUR4 cells in the absence and presence of cytoD. (Scramble – n=46/49 nuclei for DMSO/cytoD. PUR4 – n=48/60 nuclei for DMSO/cytoD from 3 independent experiments; two-way ANOVA with Tukey’s multiple comparisons test). The red bars represent the values of nuclear height obtained from the computational model. i. Example images of YAP stained cells for cells blocked with IgG control or Integrin α 5 β 1 antibody and treated with indicated pharmacological treatment for 30 minutes. Scale bar 20µm. j. Quantification of N/C YAP ratio for cells blocked with IgG control or Integrin α 5 β 1 and subjected to 30min pharmacological treatment. (IgG – n=60/65/67 cells for DMSO/bleb/cytoD. Integrin α 5 β 1 – n=103/109/109 cells for DMSO/bleb/cytoD from at least 4 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). k. Example images of mechano-reporter L_NLS-41-GFP transfected cells treated with IgG control or integrin α 5 β 1 antibody at different timepoints after treatment with cytoD. Scale bar 20µm. l. Quantification of change in N/C L_NLS-41-GFP ratio (normalized to the pre-cytoD treatment ratio) for control IgG blocked cells treated with cytoD (grey) and integrin α5β1 blocked cells treated with cytoD (blue). Solid line represents the average of all trajectories and shaded area is the standard error. IgG n=21 cells, Integrin α5β1 n=25 cells from 5 independent experiments.

Journal: bioRxiv

Article Title: Fibrillar adhesion dynamics govern the timescales of nuclear mechano-response via the vimentin cytoskeleton

doi: 10.1101/2023.11.08.566191

Figure Lengend Snippet: a. Example images of integrin-α 5 , clone snaka51 stained cells in scramble or PUR4 peptide seeded for 4 hours in presence of 500nM peptide. Scale bar 20µm, zoom 5µm. b. Percentage area under the nucleus occupied by fibrillar adhesions for scramble and PUR4 peptide. (n=74/109 cells for scramble/PUR4 from 4 independent experiments; unpaired t-test). c. Example images of YAP stained cells treated for 30min with indicated pharmacological treatment for scramble and PUR4 peptide. Scale bar 20µm. d. Quantification of N/C YAP ratio for cells in scramble or PUR4 peptide conditions treated with 30min pharmacological treatment. (Scramble – n=80/74/82 cells for DMSO/bleb/cytoD. PUR4 – n=93/118/110 cells for DMSO/bleb/cytoD from at least 3 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). e. Example images of mechano-reporter L_NLS-41-GFP transfected cells in scramble or PUR4 peptide at different time points are treatment with cytoD. Scale bar 20µm. f. Quantification of change in N/C L_NLS-41-GFP ratio (normalized to the pre-cytoD treatment ratio) for scramble (grey) and PUR4 (blue) peptide conditions treated with cytoD. Scramble n=36, PUR4 n=44 cells from 3 independent experiments. g. Example images of LaminB stained nuclei for scramble and PUR4 cells treated with DMSO control or CytoD. Yellow line indicates the position of the re-slice; sale bar 5µm. h. Quantification of nuclear height for scramble and PUR4 cells in the absence and presence of cytoD. (Scramble – n=46/49 nuclei for DMSO/cytoD. PUR4 – n=48/60 nuclei for DMSO/cytoD from 3 independent experiments; two-way ANOVA with Tukey’s multiple comparisons test). The red bars represent the values of nuclear height obtained from the computational model. i. Example images of YAP stained cells for cells blocked with IgG control or Integrin α 5 β 1 antibody and treated with indicated pharmacological treatment for 30 minutes. Scale bar 20µm. j. Quantification of N/C YAP ratio for cells blocked with IgG control or Integrin α 5 β 1 and subjected to 30min pharmacological treatment. (IgG – n=60/65/67 cells for DMSO/bleb/cytoD. Integrin α 5 β 1 – n=103/109/109 cells for DMSO/bleb/cytoD from at least 4 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). k. Example images of mechano-reporter L_NLS-41-GFP transfected cells treated with IgG control or integrin α 5 β 1 antibody at different timepoints after treatment with cytoD. Scale bar 20µm. l. Quantification of change in N/C L_NLS-41-GFP ratio (normalized to the pre-cytoD treatment ratio) for control IgG blocked cells treated with cytoD (grey) and integrin α5β1 blocked cells treated with cytoD (blue). Solid line represents the average of all trajectories and shaded area is the standard error. IgG n=21 cells, Integrin α5β1 n=25 cells from 5 independent experiments.

Article Snippet: Integrin α5, clone Snaka51 (1:300, MABT201, Millipore).

Techniques: Staining, Comparison, Transfection

a. Example of cell transfected with plectin 1f-GFP and stained with snaka51 antibody. Scale bar 20µm. b. The mean intensity of plectin 1f at snaka51 adhesions close to the cell periphery and in the central region of the cell. (n=160 adhesions from 32 cells from 3 independent experiments; Mann-Whitney test). c. Example images of vimentin morphology in cells treated with indicated pharmacological treatment for 1 hour. Black line indicates the cell outline. Scale bar 20 µm. d. The percentage area of the cell occupied for vimentin vs the percentage area under the nucleus occupied by fibrillar adhesions for 1 hour pharmacological treatment (colour coded). (Vimentin – n=59/59/59/57/47 cells for DMSO/bleb/cytoD/Y-27/latA from 3 independent experiments. Fibrillar adhesions – n=123/42/60/48/57 cells for DMSO/bleb/cytoD/Y-27/latA from at least 3 independent experiments). e. Example of vimentin stained cells in the presence of the scramble or PUR4 peptide. Black line indicates the cell periphery. Scale bar 25µm. f. The percentage area of the cell occupied by vimentin in cells cultured with scrambled or PUR4 peptide. (n=108/112 cells for scramble/PUR4 from 7 independent experiments; Mann-Whitney test). g. Schematic representation of the full length plectin 1f and the truncated plectin 1f-8 lacking the intermediate filament binding domain. h. GFP and plectin 1f-8 transfected cells stained for vimentin. Solid black line indicates the perimeter of the cell. Scale bar 25 µm. i. Quantification of the percentage area of the cell occupied by the vimentin network in GPF and plectin 1f-8 transfected cells. (n=67/112 cells for GFP/ Plectin 1f-8 from at least 5 independent experiments; Mann-Whitney test). j. Example of YAP staining for plectin 1f-8-GFP expressing cells treated for 30min with the indicated pharmacological treatment. Scale bar 20 µm. k. Quantification of N/C YAP ratio of plectin 1f-8 transfected cells after 30min pharmacological treatment. (n=120/120/132 cells for DMSO/bleb/cytoD from 5 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). l. Images of LaminB stained plectin 1f-8 transfected cells treated with DMSO and cytoD. Scale bar 5 µm. m. Quantification of nuclear height of plectin 1f-8 transfected cells treated with DMSO or cytoD. (n=48/52 nuclei for DMSO/cytoD from 3 independent experiments; unpaired two-tailed t-test). n. Scheme showing main computational model components. This includes actomyosin contractility driven by myosin motors, and a vimentin network around the nucleus anchored to the substrate via fibrillar adhesions. Fibrillar adhesions are adhesive and withstand cell/ECM forces in a dissipative way. o. Model predictions for nuclear height with anchored/unanchored vimentin (adhesions/no adhesions) and in the presence/absence of contractility. Experimental data from are shown below model predictions for comparison.

Journal: bioRxiv

Article Title: Fibrillar adhesion dynamics govern the timescales of nuclear mechano-response via the vimentin cytoskeleton

doi: 10.1101/2023.11.08.566191

Figure Lengend Snippet: a. Example of cell transfected with plectin 1f-GFP and stained with snaka51 antibody. Scale bar 20µm. b. The mean intensity of plectin 1f at snaka51 adhesions close to the cell periphery and in the central region of the cell. (n=160 adhesions from 32 cells from 3 independent experiments; Mann-Whitney test). c. Example images of vimentin morphology in cells treated with indicated pharmacological treatment for 1 hour. Black line indicates the cell outline. Scale bar 20 µm. d. The percentage area of the cell occupied for vimentin vs the percentage area under the nucleus occupied by fibrillar adhesions for 1 hour pharmacological treatment (colour coded). (Vimentin – n=59/59/59/57/47 cells for DMSO/bleb/cytoD/Y-27/latA from 3 independent experiments. Fibrillar adhesions – n=123/42/60/48/57 cells for DMSO/bleb/cytoD/Y-27/latA from at least 3 independent experiments). e. Example of vimentin stained cells in the presence of the scramble or PUR4 peptide. Black line indicates the cell periphery. Scale bar 25µm. f. The percentage area of the cell occupied by vimentin in cells cultured with scrambled or PUR4 peptide. (n=108/112 cells for scramble/PUR4 from 7 independent experiments; Mann-Whitney test). g. Schematic representation of the full length plectin 1f and the truncated plectin 1f-8 lacking the intermediate filament binding domain. h. GFP and plectin 1f-8 transfected cells stained for vimentin. Solid black line indicates the perimeter of the cell. Scale bar 25 µm. i. Quantification of the percentage area of the cell occupied by the vimentin network in GPF and plectin 1f-8 transfected cells. (n=67/112 cells for GFP/ Plectin 1f-8 from at least 5 independent experiments; Mann-Whitney test). j. Example of YAP staining for plectin 1f-8-GFP expressing cells treated for 30min with the indicated pharmacological treatment. Scale bar 20 µm. k. Quantification of N/C YAP ratio of plectin 1f-8 transfected cells after 30min pharmacological treatment. (n=120/120/132 cells for DMSO/bleb/cytoD from 5 independent experiments; Kruskal-wallis ANOVA with Dunn’s multiple comparison test). l. Images of LaminB stained plectin 1f-8 transfected cells treated with DMSO and cytoD. Scale bar 5 µm. m. Quantification of nuclear height of plectin 1f-8 transfected cells treated with DMSO or cytoD. (n=48/52 nuclei for DMSO/cytoD from 3 independent experiments; unpaired two-tailed t-test). n. Scheme showing main computational model components. This includes actomyosin contractility driven by myosin motors, and a vimentin network around the nucleus anchored to the substrate via fibrillar adhesions. Fibrillar adhesions are adhesive and withstand cell/ECM forces in a dissipative way. o. Model predictions for nuclear height with anchored/unanchored vimentin (adhesions/no adhesions) and in the presence/absence of contractility. Experimental data from are shown below model predictions for comparison.

Article Snippet: Integrin α5, clone Snaka51 (1:300, MABT201, Millipore).

Techniques: Transfection, Staining, MANN-WHITNEY, Cell Culture, Binding Assay, Expressing, Comparison, Two Tailed Test, Adhesive

(A, B, C) Representative confocal microscopy analysis of SNAKA51 + active α5 integrin (A), β3 integrin (B), and soluble rhodamine-FN (C) localization in ECs that were incubated (bottom panels) or not (top panels) for 10 min with the anti-βI domain mAb 12G10. Scale bar 20 µ m; magnification scale bar 10 µ m. (A, C) When compared with untreated control (CTL) ECs, the mFD of SNAKA51 + fibrillar adhesions or rhodamine-FN + fibrils was significantly reduced in ECs treated with mAb 12G10. Data are mean ± S.D, n ≥ 34 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (B) The incubation of cultured ECs with mAb 12G10 did not influence number, mean area, or mFD of β3 integrin + focal adhesions. Data are mean ± S.D, n ≥ 32 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test. Source data are available for this figure.

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Representative confocal microscopy analysis of SNAKA51 + active α5 integrin (A), β3 integrin (B), and soluble rhodamine-FN (C) localization in ECs that were incubated (bottom panels) or not (top panels) for 10 min with the anti-βI domain mAb 12G10. Scale bar 20 µ m; magnification scale bar 10 µ m. (A, C) When compared with untreated control (CTL) ECs, the mFD of SNAKA51 + fibrillar adhesions or rhodamine-FN + fibrils was significantly reduced in ECs treated with mAb 12G10. Data are mean ± S.D, n ≥ 34 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (B) The incubation of cultured ECs with mAb 12G10 did not influence number, mean area, or mFD of β3 integrin + focal adhesions. Data are mean ± S.D, n ≥ 32 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control, Cell Culture

Confocal immunofluorescence microscopy analysis of ECs live treated with mAb SNAKA51 ( red ) alone or in combination with mAb 12G10 ( green ). The internalization of mAb SNAKA51–bound active α5 integrins in EEA1 + early endosomes ( blue ) was quantified by Pearson correlation coefficient. Treatment with the anti-βI domain mAb 12G10 promotes mAb SNAKA51–bound active α5 integrin endocytosis. Data are mean ± SD, n ≥ 26 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. Source data are available for this figure.

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Confocal immunofluorescence microscopy analysis of ECs live treated with mAb SNAKA51 ( red ) alone or in combination with mAb 12G10 ( green ). The internalization of mAb SNAKA51–bound active α5 integrins in EEA1 + early endosomes ( blue ) was quantified by Pearson correlation coefficient. Treatment with the anti-βI domain mAb 12G10 promotes mAb SNAKA51–bound active α5 integrin endocytosis. Data are mean ± SD, n ≥ 26 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy

Left: simulation of EGFP being pulled towards the fibronectin binding site, starting when the C-terminus of the EGFP and the N-terminus of the integrin α5 are less than 1 nm apart, the linker included, leading to the formation of contacts (Movie 2). Right: simulation of a fully stretched EGFP, initially in close proximity to the fibronectin binding site, that is allowed to relax without a biasing force resulting in a spontaneous and rapid loss of contacts (<100 ns; Movie 3). The pulling process spanned 8 nm and 80 ns. The relaxation spanned 3200 ns. Contacts were calculated between EGFP and fibronectin with a cutoff of 0.6 nm.

Journal: bioRxiv

Article Title: Regulation of cell dynamics by rapid transport of integrins through the biosynthetic pathway

doi: 10.1101/2022.07.12.498931

Figure Lengend Snippet: Left: simulation of EGFP being pulled towards the fibronectin binding site, starting when the C-terminus of the EGFP and the N-terminus of the integrin α5 are less than 1 nm apart, the linker included, leading to the formation of contacts (Movie 2). Right: simulation of a fully stretched EGFP, initially in close proximity to the fibronectin binding site, that is allowed to relax without a biasing force resulting in a spontaneous and rapid loss of contacts (<100 ns; Movie 3). The pulling process spanned 8 nm and 80 ns. The relaxation spanned 3200 ns. Contacts were calculated between EGFP and fibronectin with a cutoff of 0.6 nm.

Article Snippet: Primary antibodies used: mouse anti-integrin α5 (clone SNAKA51) (MABT201, Millipore), rabbit anti-GRASP55 (HPA035274, Sigma) and rabbit anti-GRASP65 (HPA056283).

Techniques: Binding Assay

INVs are involved in recycling α5β1 integrin. (A) Bubble plot of total intensities from immunoprecipitates prepared from RPE-1 cells expressing GFP or GFP-TPD54 ( n exp = 3). (B) Bubble plot of total spectral counts from BioID2 experiments ( n replicates = 6, n exp = 2). In A and B, all TPD52-like proteins, Rabs and integrins detected in the dataset are shown. Size of bubbles is normalized to the most abundant protein detected per experiment. (C) ELISA-based quantification of integrin α5 recycling over time in siCtrl (gray line) or siTPD54-treated (blue line) RPE1 cells. **, P < 0.001; *, P < 0.05. Bars show SD. n exp = 3. (D) Representative confocal micrographs of integrin α5 recycling. RPE-1 cells (siCtrl or siTPD54 treated) were surface labeled using anti-integrin α5 (VC5) and allowed to recycle for the indicated times. The experiment was repeated three times, and similar results were also obtained using an alternative α5 antibody (SNAKA51). Scale bar, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Intracellular nanovesicles mediate α5β1 integrin trafficking during cell migration

doi: 10.1083/jcb.202009028

Figure Lengend Snippet: INVs are involved in recycling α5β1 integrin. (A) Bubble plot of total intensities from immunoprecipitates prepared from RPE-1 cells expressing GFP or GFP-TPD54 ( n exp = 3). (B) Bubble plot of total spectral counts from BioID2 experiments ( n replicates = 6, n exp = 2). In A and B, all TPD52-like proteins, Rabs and integrins detected in the dataset are shown. Size of bubbles is normalized to the most abundant protein detected per experiment. (C) ELISA-based quantification of integrin α5 recycling over time in siCtrl (gray line) or siTPD54-treated (blue line) RPE1 cells. **, P < 0.001; *, P < 0.05. Bars show SD. n exp = 3. (D) Representative confocal micrographs of integrin α5 recycling. RPE-1 cells (siCtrl or siTPD54 treated) were surface labeled using anti-integrin α5 (VC5) and allowed to recycle for the indicated times. The experiment was repeated three times, and similar results were also obtained using an alternative α5 antibody (SNAKA51). Scale bar, 10 µm.

Article Snippet: Cell surface integrins were labeled on ice for 30 min using either clone VC5 anti-human CD49e (BD PharMingen; 1:500, 555651) or clone SNAKA51 (Sigma-Aldrich; 1:500, MABT201).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Labeling

CYRIs affect integrin α5β1 trafficking. (See .) (A and B) Flow cytometry analysis of surface expression of active integrin α5, detected using the SNAKA51 antibody (A) and MT1MMP (B) comparing the control pLKO and CYRI-A/B DBKO A-673 cells. Data from three independent experiments. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. (C–E) Immunofluorescence images of the control pLKO and DBKO A-673 cells stained for active integrin α5 (magenta) and actin (cyan). The average area of integrin clusters or the number of clusters per cell are quantified in D and E. Data from three independent experiments with at least 10 cells per experiment. Each experiment is color-coded. Mean ± SD. Statistical analysis use one-way ANOVA with Tukey’s multiple comparison test. Scale bars = 20 µm. (F–H) Non-RAC1-binding mutant P16-mCherry-CYRI-A RRDD does not rescue the spreading phenotype of CYRI-B KO COS-7. Quantification of the cell spread area (G) and the Arp2/3 signal accumulating at the cell periphery (H) show that WT CYRI-A rescued these phenotypes in CYRI-B KO COS-7, while RRDD mutant did not. Data from at least 10 random fields of view in a total of three independent experiments. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. ns, P > 0.05. (I) Time sequence images of HEK293T cells coexpressing P16-GFP-CYRI-A (cyan) and mApple-integrin α5 (magenta) showing integrin α5 signal present on CYRI-A–positive vesicles. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (J–M) Immunofluorescence images of endogenous integrins α5 and β1 in A-673 cells with the P16-GFP-CYRI-A or P17-GFP-CYRI-B constructs along with actin (yellow). Graphs show the colocalization of CYRI-A, integrins, and filamentous actin (phalloidin) on the vesicles. Scale bars = 10 µm. In C, F, and J–M: DAPI for DNA.

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRIs affect integrin α5β1 trafficking. (See .) (A and B) Flow cytometry analysis of surface expression of active integrin α5, detected using the SNAKA51 antibody (A) and MT1MMP (B) comparing the control pLKO and CYRI-A/B DBKO A-673 cells. Data from three independent experiments. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. (C–E) Immunofluorescence images of the control pLKO and DBKO A-673 cells stained for active integrin α5 (magenta) and actin (cyan). The average area of integrin clusters or the number of clusters per cell are quantified in D and E. Data from three independent experiments with at least 10 cells per experiment. Each experiment is color-coded. Mean ± SD. Statistical analysis use one-way ANOVA with Tukey’s multiple comparison test. Scale bars = 20 µm. (F–H) Non-RAC1-binding mutant P16-mCherry-CYRI-A RRDD does not rescue the spreading phenotype of CYRI-B KO COS-7. Quantification of the cell spread area (G) and the Arp2/3 signal accumulating at the cell periphery (H) show that WT CYRI-A rescued these phenotypes in CYRI-B KO COS-7, while RRDD mutant did not. Data from at least 10 random fields of view in a total of three independent experiments. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. ns, P > 0.05. (I) Time sequence images of HEK293T cells coexpressing P16-GFP-CYRI-A (cyan) and mApple-integrin α5 (magenta) showing integrin α5 signal present on CYRI-A–positive vesicles. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (J–M) Immunofluorescence images of endogenous integrins α5 and β1 in A-673 cells with the P16-GFP-CYRI-A or P17-GFP-CYRI-B constructs along with actin (yellow). Graphs show the colocalization of CYRI-A, integrins, and filamentous actin (phalloidin) on the vesicles. Scale bars = 10 µm. In C, F, and J–M: DAPI for DNA.

Article Snippet: Anti–CYRI-A antibody (rabbit, Sigma-Aldrich, not in production; Western blotting), anti–CYRI-B antibody (rabbit, #HPA009076, RRID:AB_1848402; Sigma-Aldrich; Western blotting), anti-GAPDH (mouse, #MAB374, RRID:AB_2107445; Millipore; Western blotting), anti-Tubulin DM1A (mouse, #T6199, RRID:AB_477583; Sigma-Aldrich; Western blotting), anti-GST (rabbit, #2622, RRID:AB_331670; CST; Western blotting), anti-GFP (mouse, #2955, RRID:AB_1196614; CST; Western blotting), Phalloidin 488 (#A12379; Molecular Probes), anti-Integrin β 1 antibody [12G10] (rabbit, #ab30394, RRID:AB_775726; Abcam; immunofluorescent and Western blotting), recombinant anti-Integrin α 5 antibody [EPR7854] (rabbit, #ab150361, RRID:AB_2631309; Abcam; immunofluorescent and Western blotting), anti-Integrin α5 (preservative free) antibody, clone SNAKA51 (mouse, #MABT201; Millipore; immunofluorescent, internalization assay, and flow cytometry), Alexa Fluor 488 anti-human CD29 antibody TS2/16 (mouse, #303015, RRID:AB_493026; BioLegend; flow cytometry), Alexa Fluor 488 anti-mouse CD49e Antibody (rat, #103810, RRID:AB_528839; BioLegend; flow cytometry), anti-MMP-14 antibody, catalytic domain, clone LEM-2/63.1 (mouse, #MAB3329, RRID:AB_570600; Millipore; flow cytometry), donkey anti-rabbit 680-nm (#A10043, AB_2534018; Invitrogen; Western blotting), donkey anti-mouse 680-nm (#A10038, AB_2534014; Invitrogen; Western blotting), goat anti-mouse 800-nm (#SA5-35521, AB_2556774; Thermo Fisher Scientific; Western blotting), and goat anti-rabbit 800-nm (#SA5-35571, AB_2556775; Thermo Fisher Scientific; Western blotting).

Techniques: Flow Cytometry, Expressing, Control, Comparison, Immunofluorescence, Staining, Binding Assay, Mutagenesis, Sequencing, Construct

CYRI-A and CYRI-B cooperatively regulate the internalization of integrin α5β1. (See .) (A and B) Time sequence images of COS-7 cells coexpressing either P16-GFP-CYRI-A or P17-GFP-CYRI-B and mApple-tagged integrin α5. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (C–H) Colocalization analysis of P16-GFP-CYRI-A or P17-GFP-CYRI-B (cyan) with endogenous integrin α5 or β1 (magenta) and actin (orange) in COS-7 cells. Right panels (D, F, and H) are intensity graphs showing the colocalization of all three normalized signals at the cups/vesicles. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (I and J) Internalization assay of A-673 cells for active integrin α5 (SNAKA51, cyan) after 0- or 30-min incubation. (I) Representative images of the internalized signal of active integrin α5 (SNAKA51, black dots). (J) Quantification of the area of the internalized signals. Data from three independent experiments of at least 10 cells per experiment. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. Scale bar = 10 µm. (K and L) Reexpression of P16-GFP-CYRI-A in CYRI DBKO A-673 cells rescued the internalization defect in these cells compared to cells expressing only GFP. (L) Quantification of K. Data from three independent experiments of at least 10 cells per experiment. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. Scale bar = 10 µm. (M and N) CYRI DBKO A-673 reexpressing P16-GFP-CYRI-A treated with 10 µM LY294002 show reduction in the internalized active integrin α5 signal compared the untreated cells. (N) Quantification of at least 10 cells per experiment in a total of three independent experiments. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. Scale bar = 10 µm. ns, P > 0.05.

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI-A and CYRI-B cooperatively regulate the internalization of integrin α5β1. (See .) (A and B) Time sequence images of COS-7 cells coexpressing either P16-GFP-CYRI-A or P17-GFP-CYRI-B and mApple-tagged integrin α5. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (C–H) Colocalization analysis of P16-GFP-CYRI-A or P17-GFP-CYRI-B (cyan) with endogenous integrin α5 or β1 (magenta) and actin (orange) in COS-7 cells. Right panels (D, F, and H) are intensity graphs showing the colocalization of all three normalized signals at the cups/vesicles. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (I and J) Internalization assay of A-673 cells for active integrin α5 (SNAKA51, cyan) after 0- or 30-min incubation. (I) Representative images of the internalized signal of active integrin α5 (SNAKA51, black dots). (J) Quantification of the area of the internalized signals. Data from three independent experiments of at least 10 cells per experiment. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. Scale bar = 10 µm. (K and L) Reexpression of P16-GFP-CYRI-A in CYRI DBKO A-673 cells rescued the internalization defect in these cells compared to cells expressing only GFP. (L) Quantification of K. Data from three independent experiments of at least 10 cells per experiment. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. Scale bar = 10 µm. (M and N) CYRI DBKO A-673 reexpressing P16-GFP-CYRI-A treated with 10 µM LY294002 show reduction in the internalized active integrin α5 signal compared the untreated cells. (N) Quantification of at least 10 cells per experiment in a total of three independent experiments. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. Scale bar = 10 µm. ns, P > 0.05.

Techniques: Sequencing, Incubation, Comparison, Expressing

Constitutively active αvβ3 signaling does not alter α5β1 integrin localization within fibrillar adhesions. Subconfluent EV, WTβ3, or CAβ3 cells were plated onto glass coverslips for 24 hours prior to fixation. Cells were double-labeled with mAb SNAKA51 against the active α5β1 integrin (D, G, J) and rabbit anti-fibronectin antiserum (FN) (E, H, K). As a control, EV cells (A–C) were double-labeled with mAb GAL-13 against β-galactosidase (A) and R-NIS (B). Extensive colocalization (arrowheads) of α5β1 integrin and fibronectin within fibrillar adhesions was observed in all three cell lines (F, I, L). WTβ3 and CAβ3 cells (not shown) were also double-labeled with mAb GAL-13 and R-NIS with identical results as observed with EV cells. This labeling was performed three times with identical results. Scale bar: 50 μm.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Activation of αvβ3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner

doi: 10.1167/iovs.19-27171

Figure Lengend Snippet: Constitutively active αvβ3 signaling does not alter α5β1 integrin localization within fibrillar adhesions. Subconfluent EV, WTβ3, or CAβ3 cells were plated onto glass coverslips for 24 hours prior to fixation. Cells were double-labeled with mAb SNAKA51 against the active α5β1 integrin (D, G, J) and rabbit anti-fibronectin antiserum (FN) (E, H, K). As a control, EV cells (A–C) were double-labeled with mAb GAL-13 against β-galactosidase (A) and R-NIS (B). Extensive colocalization (arrowheads) of α5β1 integrin and fibronectin within fibrillar adhesions was observed in all three cell lines (F, I, L). WTβ3 and CAβ3 cells (not shown) were also double-labeled with mAb GAL-13 and R-NIS with identical results as observed with EV cells. This labeling was performed three times with identical results. Scale bar: 50 μm.

Article Snippet: The mouse anti-αvβ3 clone LM609 (cat. #MAB1976) and mouse anti-α5 integrin clone SNAKA51 (cat. #MABT201) were purchased from EMD Millipore (Burlington, MA, USA).

Techniques: Labeling, Control

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