Journal: PLoS ONE

Article Title: Colorectal Hyperplasia and Dysplasia Due to Human Carcinoembryonic Antigen (CEA) Family Member Expression in Transgenic Mice

doi: 10.1371/journal.pone.0001353

Figure Lengend Snippet: FACS profiles of WT (shaded area), CEABAC2 (dotted line) and CEABAC10 (solid line) purified colonocytes. A–B) FACS profiles of CEA detected by T84.66 mAb (A) and CEACAM6 detected by 9A6 mAb (B), show a higher cell surface expression of CEA and CEACAM6 in the CEABAC10 than CEABAC2 colonocytes, whereas wild-type (WT) colonocytes show only a background flurorescence. C) FACS profiles of integrin α5 detected by HMα5-1 mAb show increasing cell surface expression levels of integrin α5 with increasing CEA/CEACAM6 cell surface expression levels (A–B). D) FACS profiles of integrin α2 detected by HMalpha2 mAb show the same cell surface or even less expression levels of integrins α2.

Article Snippet: Rabbit polyclonal antibodies (Ab) used were: anti-CEA Ab (RbαCEA) which recognizes all human CEACAM family members; anti-integrin α5 Ab (H-104), anti-integrin β1 Ab (M-106) and anti-PARP Ab (H-250) which detects the C-terminus of PARP-1 and PARP-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-FAK Ab (Upstate Cell Signaling, Charlottesville, VA); anti-Akt Ab which detects the total level of Akt1, Akt2 and Akt3, anti-Phospho-Akt (Ser473) Ab which detects Akt1 only when phosphorylated at the Ser473 residue and also Akt2 and Akt3 when phosphorylated at the corresponding residues, anti-caspase-3 Ab which detects full length caspase-3 and the large fragment of cleaved caspase-3 (Cell Signaling Technology Inc., Beverley, MA).

Techniques: Purification, Expressing

Journal: PLoS ONE

Article Title: Colorectal Hyperplasia and Dysplasia Due to Human Carcinoembryonic Antigen (CEA) Family Member Expression in Transgenic Mice

doi: 10.1371/journal.pone.0001353

Figure Lengend Snippet: A) Immunoblots of different membrane fractions of purified colonocytes derived from sucrose density gradients show a shift in subcellular localization from high density (H) towards low density (L) membrane complexes for integrin α5, ILK, total AKT and Ser473-phosphorylated AKT (pAKT) in purified colonocytes from CEABAC2 and CEABAC10 mice relative to WT littermate mice. No such shift was observed for integrin α2, integrin β1 and FAK. GM1 is a marker for membrane raft-containing membrane fractions. Note that these immunoblots are representative of three independent experiments using at least 6 mice per mouse group. B) Antibody-mediated cross-linking of CEA and CEACAM6 with J22 mAb in the purified colonocytes accentuates the shift of ILK to the less dense membrane fractions shown in A. C) Densitometric analysis of immunoblots for AKT and pAKT shown in A. The mean densitometric ratios (the intensity of band in L divided by the one in H) from three independent experiments show a statistical significant shift of AKT and pAKT to the low density membrane complexes in the CEABAC colonocytes ( P <0.001 for AKT and P <0.005 for pAKT). Error bars = SEM (N = 3).

Article Snippet: Rabbit polyclonal antibodies (Ab) used were: anti-CEA Ab (RbαCEA) which recognizes all human CEACAM family members; anti-integrin α5 Ab (H-104), anti-integrin β1 Ab (M-106) and anti-PARP Ab (H-250) which detects the C-terminus of PARP-1 and PARP-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-FAK Ab (Upstate Cell Signaling, Charlottesville, VA); anti-Akt Ab which detects the total level of Akt1, Akt2 and Akt3, anti-Phospho-Akt (Ser473) Ab which detects Akt1 only when phosphorylated at the Ser473 residue and also Akt2 and Akt3 when phosphorylated at the corresponding residues, anti-caspase-3 Ab which detects full length caspase-3 and the large fragment of cleaved caspase-3 (Cell Signaling Technology Inc., Beverley, MA).

Techniques: Western Blot, Purification, Derivative Assay, Marker

Journal: Molecular Cancer

Article Title: Analysis of colorectal cancers in British Bangladeshi identifies early onset, frequent mucinous histotype and a high prevalence of RBFOX1 deletion

doi: 10.1186/1476-4598-12-1

Figure Lengend Snippet: Genomic alterations and expression changes of RBFOX1 in CRC. (A) Analysis of relative copy number (blue) gain and (red) loss across all cancers (CIN, MACS and BAN) shows recurrent deletions on chromosome 16p13 affecting the RBFOX1 gene. (B) Scatter plot showing RBFOX1 gene expression level as detected by quantitative real time PCR. Nineteen tumour / normal sample pairs were tested and mRNA level is expressed as ratio of RBFOX1 and HPRT concentration, [ RBFOX1 ] / [ HPRT ].

Article Snippet: Immunohistochemistry was carried out using rabbit polyclonal primary antibody against RBFOX1 (sc-135476, Santa Cruz Biotechnology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Molecular Cancer

Article Title: Analysis of colorectal cancers in British Bangladeshi identifies early onset, frequent mucinous histotype and a high prevalence of RBFOX1 deletion

doi: 10.1186/1476-4598-12-1

Figure Lengend Snippet: Immunohistochemical detection of RBFOX1. (A) Normal appearing colonic mucosa with diffuse cytoplasmic staining of most epithelium (x100). (B) Normal colon with diffuse staining of the epithelium and some stromal cells and inflammatory cells; the pericryptal sheath fibroblasts appear unstained (x200). (C) Adenocarcinoma showing absence of RBFOX1 staining in the majority of the cells lining the two-neoplastic glands in the lower half of the picture (x200). (D) Neighbouring glands show persistence (left) or reduction/ loss (right) of RBFOX1 immunoreactivity on epithelial cell cytoplasm (x200).

Article Snippet: Immunohistochemistry was carried out using rabbit polyclonal primary antibody against RBFOX1 (sc-135476, Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Staining

Journal: Molecular Cancer

Article Title: Analysis of colorectal cancers in British Bangladeshi identifies early onset, frequent mucinous histotype and a high prevalence of RBFOX1 deletion

doi: 10.1186/1476-4598-12-1

Figure Lengend Snippet: RBFOX1 mutations and single nucleotide polymorphisms found in CRC cell lines and patient tumour samples

Article Snippet: Immunohistochemistry was carried out using rabbit polyclonal primary antibody against RBFOX1 (sc-135476, Santa Cruz Biotechnology).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Reduction of lung metastasis, cell invasion, and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil-containing protein kinase pathway

doi: 10.1186/1756-9966-29-127

Figure Lengend Snippet: Effect of statins on B16BL6 cell adhesion to ECM components . B16BL6 cells, which had been treated with 0.05 μM fluvastatin or 0.1 μM simvastatin for 3 d, were incubated with (A) type I collagen-, (B) type IV collagen-, (C) fibronectin-, or (D) laminin-coated plates for 30 min at 37°C in an atmosphere containing 5% CO 2 . The results are representative of 5 independent experiments. (E) Image showing the results of RT-PCR analysis of integrins mRNA. B16BL6 cells were treated with 0.05 μM fluvastatin or 0.1 μM simvastatin. After 3 d, equal amounts of RNA were reverse-transcribed to generate cDNA, which was used for PCR analysis of integrins mRNA expression in B16BL6 cells. (E) Image showing western blot of the integrin α 2 , integrin α 4 , and integrin α 5 proteins. Whole-cell lysates were generated and immunoblotted with antibodies against integrin α 2 , integrin α 4 , integrin α 5 , and β-actin (internal standard).

Article Snippet: Inc., California, USA), anti-α 4 integrin antibody (SantaCruz Biotechnology, CA, USA), anti-α 5 integrin antibody (SantaCruz Biotechnology), and anti-Rho antibody (Upstate Biology, Charlottesville, VA, USA).

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

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Diarrheagenic E. coli bind to integrin α 5 β 1. (a) EAEC, EHEC, and ETEC were added to integrin α 5 β 1- and BSA-coated wells and bacterial binding was detected by ELISA, using anti-O44, anti-O157, and anti-O78 serum, respectively. (b) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells. EAEC binding was detected by ELISA, using anti-O44 serum. *Significantly different between treatments ( P < 0.05).

Article Snippet: Epithelial cells, grown in 96-well plates, were incubated for 30 min with 100 μ L/well of DMEM-F12 only or supplemented with fibronectin (1 μ g/well), antibodies anti-integrin α 5 β 1 that block the interaction with RGD site in fibronectin (Abcam) or anti-integrin α 5 (Santa Cruz Biotechnology).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Fibronectin/integrin α 5 β 1 complex increases the adhesion of EAEC strain 042. (a) Integrin α 5 β 1- and BSA-coated wells were incubated with increasing concentrations of fibronectin (25, 50, and 100 ng) or (b) with 100 ng of fibronectin (Fn) for 15, 30, or 60 min. Bound fibronectin was detected by ELISA, using anti-fibronectin antibodies. (c) EAEC 042 was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin. (d) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells incubated with fibronectin. EAEC binding was detected by ELISA using anti-O44 serum. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05).

Article Snippet: Epithelial cells, grown in 96-well plates, were incubated for 30 min with 100 μ L/well of DMEM-F12 only or supplemented with fibronectin (1 μ g/well), antibodies anti-integrin α 5 β 1 that block the interaction with RGD site in fibronectin (Abcam) or anti-integrin α 5 (Santa Cruz Biotechnology).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: AafA binding to fibronectin/integrin α 5 β 1 complex. (a) AafA- dsc protein was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin (Fn). AafA- dsc binding was detected by ELISA using anti-AafA antibodies. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05). (b) AafA- dsc and fibronectin or (c) AafA- dsc , fibronectin, and integrin α 5 β 1 were mixed and the complex formed was added to control column or a column with anti-AafA antibodies for coimmunoprecipitation analysis. The eluted fraction obtained from control column (E1 and E3) or a column with anti-AafA antibodies (E2 and E4) was analyzed by Dot blot, using anti-AafA, anti-integrin α 5, and anti-fibronectin antibodies. The resulting autoradiography was scanned and signal intensity was quantified by UN-SCAN-IT 6.1 software. One representative experiment of three independent experiments with similar result is shown.

Article Snippet: Epithelial cells, grown in 96-well plates, were incubated for 30 min with 100 μ L/well of DMEM-F12 only or supplemented with fibronectin (1 μ g/well), antibodies anti-integrin α 5 β 1 that block the interaction with RGD site in fibronectin (Abcam) or anti-integrin α 5 (Santa Cruz Biotechnology).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Dot Blot, Autoradiography, Software

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Adhesion of EAEC strain 042 to T84 cells. T84 cells preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn), Fn and anti-integrin α 5 β 1 (RGD), or anti-integrin α 5 β 1 ( α 5) were infected with EAEC strain 042. The number of adherent bacteria was determined by colony forming unit counts (CFU). *Significantly different between treatments ( P < 0.05).

Article Snippet: Epithelial cells, grown in 96-well plates, were incubated for 30 min with 100 μ L/well of DMEM-F12 only or supplemented with fibronectin (1 μ g/well), antibodies anti-integrin α 5 β 1 that block the interaction with RGD site in fibronectin (Abcam) or anti-integrin α 5 (Santa Cruz Biotechnology).

Techniques: Infection

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Integrin α 5 expression knockdown reduces EAEC strain 042 fibronectin-mediated binding to epithelial cells. HEp-2 cells nontransfected and transfected with scrambled or integrin α 5 shRNA were preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn) and then infected with EAEC strain 042. Numbers of adherent bacteria were determined by colony forming unit counts (CFU). *Significantly different compared to control treatment ( P < 0.05).

Article Snippet: Epithelial cells, grown in 96-well plates, were incubated for 30 min with 100 μ L/well of DMEM-F12 only or supplemented with fibronectin (1 μ g/well), antibodies anti-integrin α 5 β 1 that block the interaction with RGD site in fibronectin (Abcam) or anti-integrin α 5 (Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Transfection, shRNA, Infection

Journal: Breast Cancer Research : BCR

Article Title: Dissecting the transcriptional networks underlying breast cancer: NR4A1 reduces the migration of normal and breast cancer cell lines

doi: 10.1186/bcr2610

Figure Lengend Snippet: Effect of NR4A1 on cell adhesion and integrin expression . (a) Control or MCF10A-NR4A1 cells were plated on wells coated with fibronectin, collagen type I, vitronectin or laminin I. Attached cells were fixed and stained with crystal violet. MCF10A-NR4A1 cells show increased adhesion to fibronectin and decreased adhesion to collagen type I compared with control cells, while both cell lines show similar adhesion levels to vitronectin and laminin I (mean ± standard deviation of six wells, * P < 0.001). (b) MCF10A-NR4A1 cells or control cells were analysed by flow cytometry for the cell surface expression of the integrins depicted. MCF10A-NR4A1 cells have increased levels of α 5 and β 4 integrins.

Article Snippet: Antibodies against α 5 integrin (P1D6) and α 6 integrin (GOH3) were from Santa Cruz.

Techniques: Expressing, Staining, Standard Deviation, Flow Cytometry

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0080026

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

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

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

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0080026

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

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

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

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0080026

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

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

Techniques: Activity Assay

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0080026

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

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

Techniques: Standard Deviation, Migration, Incubation