Journal: Nature Communications

Article Title: Control cell migration by engineering integrin ligand assembly

doi: 10.1038/s41467-022-32686-2

Figure Lengend Snippet: a Schematic illustration of precise control of integrin ligand presentation on nanofilaments via peptide assembly. b TEM images of nanofilaments obtained via molecular self-assembly and co-assembly of FFFIKLLI (100 μM) and FFF at various ratios, and the estimated molecular packing structures. IKLLI motif is presented in blue and FFF motif is presented in pink. The scale bars represent 200 nm. Three independent experiments were performed. c Zoom-in SEM images (false color) of HuH-7 cell edge and apical membrane after 3-day incubations. FFFIKLLI was maintained at a concentration of 100 μM. The cell body is highlighted in pink, while the nanofilaments are highlighted in blue. The scale bars represent 300 nm.

Article Snippet: Integrin β1 shRNA plasmid (#sc-29375-SH), Integrin α3 shRNA plasmid (#sc-35684-SH), Integrin α6 shRNA plasmid (#sc-43129-SH), and control shRNA plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology for knockdown of the target integrins.

Techniques: Control, Membrane, Concentration Assay

Journal: Nature Communications

Article Title: Control cell migration by engineering integrin ligand assembly

doi: 10.1038/s41467-022-32686-2

Figure Lengend Snippet: a Time-lapse series showing actin cytoskeleton (grey) and paxillin (green) in HuH-7 cells expressing mRuby-Lifeact-7 and mGFP-paxillin upon the treatment of FFFIKLLI (100 μM) for 12 hr. Scale bars represent 2 μm. Three independent experiments were performed. b F-actin phalloidin staining (magenta), integrin β1 (cyan) and paxillin (yellow) immunofluorescence in HuH-7 cell after 12 h treatment of FFFIKLLI (100 μM). Scale bar represents 2 μm. c Fluorescence intensity distribution profile of integrin β1, paxillin, and actin cytoskeleton along the yellow line on merged image of b . d 12 h time course Rac1 activity of HuH-7 cells with or without the treatment of FFFIKLLI (100 μM). Rac1 activity was measured by FRET. n = 6 cells (Ctrl) or 5 cells (FFFIKLLI). Symbols represent the mean FRET/CFP emission ratio ± s.d. e Representative FRET/CFP ratio images of HuH-7 cells expressing RaichuEV-Rac1 with or without the treatment of FFFIKLLI (100 μM) at the indicated time points and coded according to a pseudo-color scale, which ranges from yellow to purple with an increase in Rac1 activity. Scale bars represent 20 μm. Source numerical data are available in source data.

Article Snippet: Integrin β1 shRNA plasmid (#sc-29375-SH), Integrin α3 shRNA plasmid (#sc-35684-SH), Integrin α6 shRNA plasmid (#sc-43129-SH), and control shRNA plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology for knockdown of the target integrins.

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence, Activity Assay

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: Matrix Biology

Article Title: Basement membrane ligands initiate distinct signalling networks to direct cell shape

doi: 10.1016/j.matbio.2020.02.005

Figure Lengend Snippet: Adhesion to basement membrane ligand determines cellular morphology. (A) Podocytes were allowed to attach to plates coated with 0–10 μg/ml of matrix substrate for 30 min in serum-free media. Non-attached cells were removed by washing with PBS, and the percent of added cells attached to the substrate was quantified by crystal violet staining. (B) Podocytes attached to 5 μg/ml of matrix substrate for 240 min in serum-free media; collagen IV, laminin 511 and laminin 521. Cell spread area was calculated using phase-contrast live cell imaging. Measurements of cell area were extracted using Fiji ImageJ. (C–J) Podocytes were spread on 5 μg/ml of matrix substrate for 210 min in serum-free media. (C) Phalloidin staining of podocytes highlights distinct cellular shapes and actin structures within podocytes adhered to collagen IV compared with laminin. The colour-coded shape outlines indicate representative protrusive activities at 5-min intervals recorded between 180 and 280 min of cell spreading. (D) Podocytes circularity assessment when attached to collagen IV compared with laminin. Circularity was calculated using Fiji ImageJ. (E) Cell morphology was assessed using Fiji ImageJ; cells were manually categorised as rounded elongated or containing multiple protrusions. Podocytes had more elongated shapes and also produced more pseudopodial protrusions when spread on laminin compared with collagen IV. (F) Integrin β1 foci were larger in podocytes spread on collagen IV compared with podocytes spread on laminin. (G–H) Ratio imaging demonstrated differential localization of (G) vinculin and (H) talin to integrin adhesion complexes formed on collagen IV and laminin. (I–J) Immunofluorescence of Integrin β1 and (I) vinculin and (J) talin in podocytes spread on collagen IV or laminin. For attachment and spreading assays, experiments were performed four times. For immunofluorescence and morphology-determining experiments, 20–40 cells were measured per experiment and each experiment was performed four times. (I, J) Scale bar represents 10 μm; ∗∗∗∗, p < 0.0001; NS, not significant; LAM511, laminin-511; LAM521, laminin-521; COL4, collagen IV. Bar and line graph measurements are shown as mean ± standard deviation. Box plots indicate 25th and 75th percentiles (lower and upper bounds, respectively), 1.5 × interquartile range (whiskers) and median (black line). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Single-sorted cells were allowed to expand and deletion of integrin α3 was verified by immunoblotting using a goat anti-human integrin α3 antibody (I-19; Santa Cruz Biotechnology).

Techniques: Membrane, Staining, Live Cell Imaging, Produced, Imaging, Immunofluorescence, Standard Deviation

Journal: Matrix Biology

Article Title: Basement membrane ligands initiate distinct signalling networks to direct cell shape

doi: 10.1016/j.matbio.2020.02.005

Figure Lengend Snippet: Signalling response to basement membrane (BM) ligands. (A–D) Canonical integrin signalling assayed by Western blot of total cell lysates from podocytes spreading on BM ligands over a 210-min time course of cells spread on 5 μg/ml of matrix substrate. (A–D) Podocyte P-Erk (T202/Y204), P-Akt (S473), P-Src (T416) and P-FAK (Y397) levels at 0, 15, 30 and 210 min of cell spreading onto either type IV collagen or laminin 511. Band intensity was calculated using integrated density in Fiji imageJ. (E) Immunofluorescence and ratio imaging quantification of P-FAK (Y397) and Integrin β1 in podocytes after 210 min of cell spreading onto BM ligands. For these experiments 20 cells were measured per experiment and each experiment was performed four times (F) GST-PAK pull down of active Rac1 assayed in podocytes spreading in serum-free media on 5 μg/ml of matrix substrate over a 210-min time course (0, 15, 30, 105 and 210 min of cell spreading). All bar graph measurements are shown as mean ± standard deviation. Box plots indicate 25th and 75th percentiles (lower and upper bounds, respectively), 1.5 × interquartile range (whiskers) and median (black line). Scale bar in (E) represents 10 μm; ∗∗∗∗, p < 0.0001; NS, not significant; LAM511, laminin-511; COL4, collagen IV.

Article Snippet: Single-sorted cells were allowed to expand and deletion of integrin α3 was verified by immunoblotting using a goat anti-human integrin α3 antibody (I-19; Santa Cruz Biotechnology).

Techniques: Membrane, Western Blot, Immunofluorescence, Imaging, Standard Deviation

Journal: Matrix Biology

Article Title: Basement membrane ligands initiate distinct signalling networks to direct cell shape

doi: 10.1016/j.matbio.2020.02.005

Figure Lengend Snippet: The adhesome is matrix ligand dependent. (A) Principal component analysis of mass spectrometry data from podocytes. Circles represent biological replicates. (B) Unsupervised hierarchical clustering analysis of integrin adhesion complexes (IACs). Clustering was performed on the basis of Euclidian distance. The heat map displays row z-score, plotted in hot–cold colour scale, with hot (red-orange) indicating high abundance and cold (green-blue) indicating low abundance. Summary protein profiles across conditions are displayed. Grey lines show individual protein profiles in the highlighted cluster and the central coloured line represents the mean protein abundance of the cluster. Associated Gene Ontology enrichment maps demonstrate enriched biological processes. The size of the nodes (circles) represents the number of proteins, the grey lines (edges) between the nodes represents overlap of proteins within biological process terms. The intensity (opacity) of colouring represents the p value of the enrichment. (C) Protein-protein interaction network constructed from known integrin adhesion components that were identified by mass spectrometry. Proteins are sorted into different categories. Edges represent protein-protein interactions, nodes represent proteins. Solid edges represent reported protein-protein interactions detected using multiple different techniques, dashed lines represent lower confidence interactions detected with single techniques. Coloured nodes represent those with significantly different abundance in laminin (511 and 521 combined) IACs compared with collagen IV IACs. (D) Immunofluorescence of filamin A and integrin β1 in cells spread on matrix-ligand for 210 min. (E) Immunofluorescence of PKCα and integrin β1 in cells spread on matrix-ligand for 210 min. (F–J) Ratio imaging analysis of integrin adhesion components identified as either laminin or collagen IV enriched by MS. For immunofluorescence and morphology-determining experiments, 20–40 cells were measured per experiment and each experiment was performed four times. Box plots indicate 25th and 75th percentiles (lower and upper bounds, respectively), 1.5 × interquartile range (whiskers) and median (black line). Scale bar in (D, E) represents 10 μm; ∗∗∗∗, p < 0.0001; NS, not significant; LAM511, laminin-511; COL4, collagen IV; ApoT, apotransferrin. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Single-sorted cells were allowed to expand and deletion of integrin α3 was verified by immunoblotting using a goat anti-human integrin α3 antibody (I-19; Santa Cruz Biotechnology).

Techniques: Mass Spectrometry, Quantitative Proteomics, Construct, Protein-Protein interactions, Immunofluorescence, Imaging

Journal: Matrix Biology

Article Title: Basement membrane ligands initiate distinct signalling networks to direct cell shape

doi: 10.1016/j.matbio.2020.02.005

Figure Lengend Snippet: Modulating PKC alters cell morphology and signalling. (A) Western blot demonstrates PKC activity in both total cell lysates and integrin adhesion complexes from podocytes spread onto collagen IV or laminin for 210 min. (B) The colour-coded shape outlines indicate representative protrusive activities at 5-min intervals recorded over 100-min period. For measurements of protrusive activity live cell imaging was performed between 180 and 280 min of cell spreading. Podocytes were treated with either GӦ6976 or PMA to suppress or induce PKC activity respectively during cell spreading on matrix ligand. (C,D) PKC modulation impacts podocyte morphology and pseudopodial projection formation. (E,F) PKCα localization to adhesions is controlled through ligand specificity. Podocytes were spread on circles and lines coated with collagen IV or laminin for 3.5 h in serum-free media and stained for PKCα and paxillin and ratiometric imaging was performed. Scale bar represents 20 μm. Data are pooled from three independent experiments. GӦ6976 induced inhibition of PKCa localization to FA was significant for collagen circles, lines and laminin lines. For ratiometric imaging 20–40 cells were analysed per experiment and each experiment was performed four times. All bar graph measurements are shown as mean ± standard deviation. Box plots indicate 25th and 75th percentiles (lower and upper bounds, respectively), 1.5 × interquartile range (whiskers) and median (black line). Scale bar in (e) represents 5 μm; ∗∗∗∗, p < 0.0001; NS, not significant; LAM511, laminin-511; COL4, collagen IV. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Single-sorted cells were allowed to expand and deletion of integrin α3 was verified by immunoblotting using a goat anti-human integrin α3 antibody (I-19; Santa Cruz Biotechnology).

Techniques: Western Blot, Activity Assay, Live Cell Imaging, Staining, Imaging, Inhibition, Standard Deviation

Journal: Matrix Biology

Article Title: Basement membrane ligands initiate distinct signalling networks to direct cell shape

doi: 10.1016/j.matbio.2020.02.005

Figure Lengend Snippet: Integrin α3 and PKC are involved in matrix interpretation. (A) Deletion of ITGA3 from HEK293T cells using two different CRISPR-Cas9 guide RNAs. Ctrl, scrambled guide RNA. Lane 1, molecular weight marker; lane 2, control guide RNA; lane 3, ITGA3 guide RNA 1 targeted cells prior to fluorescent activated cell sorting; lane 4, ITGA3 guide RNA 2 targeted cells prior to fluorescent activated cell sorting; lane 5, ITGA3 guide RNA 1 targeted cells after fluorescent activated cell sorting; lane 6, ITGA3 guide RNA 1 targeted cells after fluorescent activated cell sorting. (B) Immunofluorescence of ITGA3-KO cells spread onto matrix ligand for 210 min. (C) Western blot demonstrates PKC activity in integrin adhesion complexes from ITGA3-KO cells spread onto matrix ligand for 210 min. (D–F) Deletion of ITGA3 impacts cell morphology and pseudopodial projection formation. (G) GST-PAK pull down of active Rac1 assayed in podocytes spreading on BM ligands over a 210-min time course. All bar graph measurements are shown as mean ± standard deviation. For immunofluorescence and morphology-determining experiments, 20–40 cells were measured per experiment and each experiment was performed four times. All bar graph measurements are shown as mean ± standard deviation. Box plots indicate 25th and 75th percentiles (lower and upper bounds, respectively), 1.5 × interquartile range (whiskers) and median (black line). Scale bar in (f) represents 10 μm; ∗∗∗∗, p < 0.0001; NS, not significant; LAM511, laminin-511; COL4, collagen IV.

Article Snippet: Single-sorted cells were allowed to expand and deletion of integrin α3 was verified by immunoblotting using a goat anti-human integrin α3 antibody (I-19; Santa Cruz Biotechnology).

Techniques: CRISPR, Molecular Weight, Marker, Control, FACS, Immunofluorescence, Western Blot, Activity Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: ( A ) CD46 (green), PNA lectin (red). ( B ) β1 integrin (green), PNA lectin (red). The first column represents a schematic illustration of ( A ) CD46, ( B ) β1 integrin localization in the acrosome intact sperm (line I) and in sperm during the AR progress. ( A ) CD46 detection in intact acrosomal membranes (line I), and the residual outer acrosomal membrane (line II) (see the green arrow), the inner acrosomal membrane begins to emerge (see the red arrow). CD46 relocation progress during the AR is seen across the apical equatorial segment towards the whole equatorial segment and the whole sperm head. ( B ) β1 integrin is relocated across the apical equatorial segment towards the whole equatorial segment and the whole sperm head. In contrast to CD46, the residual acrosome cap and the apical equatorial segments were not detected. Scale bar represents 2 μm.

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Membrane

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: Individual bars denote the percentage distribution of CD46 and β1 integrin staining patterns among individual times of capacitation and induced AR. Error bars denote SEM. AC – Acrosome Cap, rAC – residual Acrosome Cap, aES – apical Equatorial Segment, ES – Equatorial Segment, WSH – Whole Sperm Head. C – time of the capacitation, AR – time of the induced acrosome reaction.

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Staining

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: ( A ) Coloured lines with error bars represent the relative fluorescent intensities of β1 integrin (red) and CD46 (green) among the individual segments of the sperm head in 10 sperm with an intact acrosome. Horizontal coloured lines represent the arithmetic means of the fluorescent intensities for β1 integrin (red) and CD46 (green). ( B ) Coloured lines with error bars represent the relative fluorescent intensities of β1 integrin (red) and CD46 (green) among the individual segments of the sperm head in 10 acrosome reacted sperm. ( C ) Statistical comparison of the relative fluorescent intensities of β1 integrin (red) and CD46 (green) among individual segments of the sperm head between the acrosome-intact and acrosome-reacted sperm. Horizontal coloured lines represent the arithmetic means of the fluorescent intensities for β1 integrin (red) and CD46 (green). Error bars denote SEM. AC – Acrosome Cap, ES – Equatorial Segment, PAR – Post-Acrosomal Region. p value equal or lower than 0.05 was considered to be significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Comparison

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: Interactions of CD46 and β1 integrin in C57BL/6 spermatozoa determined by Duolink proximity ligation assay. ( A ) freshly released sperm, ( B ) sperm during the induced AR. Positive control (α tubulin/ β tubulin), negative control (β1 integrin/ α tubulin). The small pictures in the corners represent the immunofluorescent dual staining of CD46 and β1 integrin, CD46 (green), β1 integrin (red). Scale bar represents 4 μm.

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Proximity Ligation Assay, Positive Control, Negative Control, Staining

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: ( A ) (line I) CD46 (green) is locked over the acrosome vesicle and β1 integrin (red) is confined to both the plasma and the outer acrosomal membrane with prominent labelling of the perforatorium (see the red arrow); (line II) the onset of AR with the membrane vesiculation is visible, as well as the visibility of CD46 and β1 integrin within the acrosome and plasma membranes (see green and red arrows); (line III) the loss of the acrosome including the CD46 and β1 integrin signal is visible. Relocation of CD46 and β1 integrin over the equatorial segment in patchy clusters can be recognised; (line IV) CD46 is localized through the inner acrosome membrane (see the green arrow), the equatorial segment and over the post-acrosome region. β1 integrin shows a similar pattern to CD46 and also remains localized in the perforatorium (see the red arrow). ( B ) (line I) α6 (green) and β1 (red) integrins occupy different regions of the intact sperm head, except the same localization in the plasma membrane over the acrosome and the hook. α6 integrin is continues to be spread over the equatorial segment, even prior to AR (see the green arrows). β1 integrin is further present in the outer acrosomal membrane (when compared with the CD46 dual staining in AI) and perforatorium (see the red arrows). (line II) The α3 (red) integrin pattern is clearly different to the α6 (green) subunit, but remarkably similar to the one of β1 (red, line I) in the acrosome region. α3 is expressed on in the outer acrosomal membrane and the plasma membrane over the acrosome. (line III) The differences in the localization of the α3 (red) integrin and CD46 (green) are visible. The α3 integrin subunit is detectable on the plasma (see also for detail) and the outer acrosomal membrane, when CD46 is defined strictly to the acrosomal membranes only. DAPI (blue); Scale bar represents 1 μm.

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Clinical Proteomics, Membrane, Staining

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: (line I and III) Actin (red) fills the apical acrosome and equatorial segment of an intact sperm head, it copies the plasma membrane and overlays the CD46 (green, line I) labelled acrosome in a thin line pattern (see red arrow). (line III) β1 integrin and actin display a similar localization in the apical acrosome cap region (plasma and acrosomal membrane). The perforatorium is filled with actin, but clearly marked with the β1 integrin. (line II and IV) Actin (red) is confined to the equatorial and postacrosomal segment in the acrosome reacted sperm. CD46 (green, line II) and β1 (green, line IV) relocation progress during the AR is visible. DAPI (blue); Scale bar represents 1 μm.

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Clinical Proteomics, Membrane

Journal: Scientific Reports

Article Title: Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

doi: 10.1038/srep33714

Figure Lengend Snippet: ( A ) SIM data show the localization of CD46 (green) on the inner and outer acrosomal membrane and β1 integrin (red) on the plasma and outer acrosomal and plasma membrane of the acrosomal area. Scale bar represents 1 μm. ( B ) SIM super-resolution image analysed by Huygens software, showing the colocalization area (yellow) of selected proteins in the outer acrosomal membrane. The colocalization map is based on Pearson’s correlation coefficient. Scale bar represents 1 μm. DAPI (blue).

Article Snippet: The nitrocellulose membranes were incubated with the primary antibody for 1.5 h, washed six times for 5 min with a wash solution (0.05% Tween-20 in PBS) and incubated with a peroxidase-conjugated secondary antibody for 1 h. Proteins were identified as follows; CD46: rat monoclonal antibody (MM10, Hycult Biotech), 1:100 followed by donkey anti rat IgG antibody conjugated to HRP (170 5046, Bio-Rad), 1:10000; β1 integrin: rabbit polyclonal antibody (M-106: sc-8978 Santa Cruz Biotechnology) 1:20, followed by a peroxidase goat anti rabbit IgG (170–6515 BioRad), 1:3000; α3 integrin: primary rabbit polyclonal antibody (H-43): sc-28665 Santa Cruz Biotechnology diluted 1:20 and peroxidase goat anti rabbit IgG secondary antibody conjugated to HRP (170–6515 BioRad), 1:3000; α6 integrin: mouse monoclonal antibody (F-6): sc-374057 Santa Cruz Biotechnology 1:20, followed by a goat anti mouse IgG conjugated to HRP (170–6516 BioRad) 1:5000.

Techniques: Membrane, Clinical Proteomics, Software

Journal: Scientific Reports

Article Title: Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer

doi: 10.1038/s41598-019-57287-w

Figure Lengend Snippet: Immunoprecipitation and immunodetection by Western blot of ( A ) integrin α3 and ( B ) CD26 antigen with novel mAbs KU44.22B and KU44.13A using lysates from CaOV-3 ovarian cancer cells and AsPC-1 pancreatic cancer cells respectively. Left panel: Immunoprecipitation was performed with novel mAbs ( A ) KU44.22B and ( B ) KU44.13A (5 µg) using sheep anti-mouse dynabeads. Protein bands around ~140 KDa and ~ 260KDa were immunoprecipitated with mAb KU44.22B (A; left panel) and ~110 KDa by mAb KU44.13A (B; left panel) respectively and stained with SimplyBlue™ SafeStain. The ~50/25 KDa bands represent heavy and light chains of the anti-mouse antibody. *( B ) left panel corresponds to a cropped gel; vertically sliced images of juxtaposed lanes that were non-adjacent in the gel have a clear separation delineating the boundary between the gels. Middle panel: Integrin α3 and CD26 antigen were immunoprecipitated with mAbs ( A ) KU44.22B and ( B ) KU44.13A (5 µg) respectively, and probed with the same antibody (30 µg/ml). Target antigens were not immunodetected with either of the mAbs. Right panel: Integrin α3 and CD26 antigen were immunoprecipitated with mAbs ( A ) KU44.22B and ( B ) KU44.13A respectively (5 µg) or commercial anti-integrin α3 and anti-CD26 antibodies (2 µg) and immunodetected with commercial mAbs sc-374242 and ab89398 as described in Methods. Immunodetection of target antigens immunoprecipitated by novel mAbs and probed with commercial mAbs confirmed the target identity. MW: molecular weight marker.

Article Snippet: In conclusion, in this study, we reported the production of two novel monoclonal antibodies against integrin α3 and CD26 on human pancreatic cancer cells, using the liver metastasis pancreatic cancer cell line CFPAC-1 as immunogen.

Techniques: Immunoprecipitation, Immunodetection, Western Blot, Staining, Molecular Weight, Marker

Journal: Scientific Reports

Article Title: Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer

doi: 10.1038/s41598-019-57287-w

Figure Lengend Snippet: Identification of proteins recognised by novel mAbs KU44.13A and KU44.22B by mass spectrometry.

Article Snippet: In conclusion, in this study, we reported the production of two novel monoclonal antibodies against integrin α3 and CD26 on human pancreatic cancer cells, using the liver metastasis pancreatic cancer cell line CFPAC-1 as immunogen.

Techniques:

Journal:

Article Title: A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes

doi: 10.1073/pnas.1031602100

Figure Lengend Snippet: Electron microscopy of SNSs. (A–C) Presynaptic endings containing vesicles and postsynaptic structures (arrows) with postsynaptic densities and scattered polysomes. (Scale bar = 0.5 μm.) (B and C) Larger magnification of SNSs with or without 0.01% Triton X-100 treatment, respectively. (Scale bar = 0.5 μm.) (D) Label-fracture replica showing the ImmunoGold (Aurion, Wageningen, The Netherlands) labeling of the α3 integrin receptor subunit (circles) in a postsynaptic membrane, E-face. (Calibration bar = 0.4 μm.) (E) Electron micrograph showing the ImmunoGold labeling of the α3 integrin subunit in unfractured SNSs. The round black areas observed in the micrograph correspond to presynaptic terminals to which remain attached postsynaptic membranes showing heavy labeling for the α3 integrin subunit (black dots). (Calibration bar = 0.4 μm.)

Article Snippet: Fig. D shows that Arc mRNA is compartmentalized in the cytosolic and translocates in the polyribosomal fraction after reelin addition. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Electron microscopy of SNSs. ( A – C ) Presynaptic endings containing vesicles and postsynaptic structures (arrows) with postsynaptic densities and scattered polysomes. (Scale bar = 0.5 μm.) (B and C) Larger magnification of SNSs with or without 0.01% Triton X-100 treatment, respectively. (Scale bar = 0.5 μm.) ( D ) Label-fracture replica showing the ImmunoGold (Aurion, Wageningen, The Netherlands) labeling of the α3 integrin receptor subunit (circles) in a postsynaptic membrane, E-face. (Calibration bar = 0.4 μm.) ( E ) Electron micrograph showing the ImmunoGold labeling of the α3 integrin subunit in unfractured SNSs.

Techniques: Electron Microscopy, Labeling, Membrane

Journal:

Article Title: A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes

doi: 10.1073/pnas.1031602100

Figure Lengend Snippet: Biochemical characteristics of SNSs. (A) Western immunoblot with antibodies specific for reelin, 400- and 320-kDa (A1); postsynaptic density protein, 95-kDa (A2); and synaptophysin, 35-kDa proteins (A3). (B) Western immunoblot with α3 or β1 integrin receptor subunit-specific antibodies in 0.01% Triton X-100-treated SNSs. (C) Western immunoblot with reelin-specific (G10) antibody of SNSs before and after treatment with 0.01% Triton X-100. (D) RT-PCR of Arc mRNA in SNSs incubated without (lanes a and c) or with (lanes b and d) reelin (30 pM/45 min at 22°C and 30 min at 37°C). Total SNS (I) and polyribosome (II) Arc mRNA. (E) S.B. of [3H]flunitrazepam (2 nM) to SNS, myelin (My), and mitochondria (Mt) fractions. Each bar is the mean ± SE of triplicate assays. (F) S.B. of [125I]echistatin (50 fM) to the samples described in E. Each bar is the mean ± SE of triplicate assays.

Article Snippet: Fig. D shows that Arc mRNA is compartmentalized in the cytosolic and translocates in the polyribosomal fraction after reelin addition. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Electron microscopy of SNSs. ( A – C ) Presynaptic endings containing vesicles and postsynaptic structures (arrows) with postsynaptic densities and scattered polysomes. (Scale bar = 0.5 μm.) (B and C) Larger magnification of SNSs with or without 0.01% Triton X-100 treatment, respectively. (Scale bar = 0.5 μm.) ( D ) Label-fracture replica showing the ImmunoGold (Aurion, Wageningen, The Netherlands) labeling of the α3 integrin receptor subunit (circles) in a postsynaptic membrane, E-face. (Calibration bar = 0.4 μm.) ( E ) Electron micrograph showing the ImmunoGold labeling of the α3 integrin subunit in unfractured SNSs.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal:

Article Title: A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes

doi: 10.1073/pnas.1031602100

Figure Lengend Snippet: Competition between echistatin and reelin binding to integrin receptors. (A) S.B. [125I]echistatin to SNS membranes treated with 0.01% Triton X-100 to remove native reelin. (Inset) Scatchard analysis of the data. (B) Recombinant reelin dose-dependent displacement of [125I]echistatin binding to integrin receptors. Culture medium from mock transfected cells [(−)reelin] was used as control. Each data point is the average of triplicate measurements. Experiments were repeated three to five times, yielding similar results.

Article Snippet: Fig. D shows that Arc mRNA is compartmentalized in the cytosolic and translocates in the polyribosomal fraction after reelin addition. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 Electron microscopy of SNSs. ( A – C ) Presynaptic endings containing vesicles and postsynaptic structures (arrows) with postsynaptic densities and scattered polysomes. (Scale bar = 0.5 μm.) (B and C) Larger magnification of SNSs with or without 0.01% Triton X-100 treatment, respectively. (Scale bar = 0.5 μm.) ( D ) Label-fracture replica showing the ImmunoGold (Aurion, Wageningen, The Netherlands) labeling of the α3 integrin receptor subunit (circles) in a postsynaptic membrane, E-face. (Calibration bar = 0.4 μm.) ( E ) Electron micrograph showing the ImmunoGold labeling of the α3 integrin subunit in unfractured SNSs.

Techniques: Binding Assay, Recombinant, Transfection, Control