Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: Conserved residues in the C-terminal region of kindlin-2 support αIIbβ3 activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K2 and Fit1, the K2 homolog of D. melanogaster. B and C, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused FL, wild-type K2 or its mutants and DsRed talin-H, and activation of αIIbβ3 integrin was determined at 24 h by flow cytometry by staining the cells with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S1.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A, HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars, S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B, HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars, S.D. (p < 0.001). C, mouse RAW 264.7 cells were transiently transfected with plasmids encoding EGFP alone or EGFP-K2 constructs. The transfected cells were stained with 9EG7 monoclonal antibody to assess β1 integrin activation. Flow cytometry was used to measure 9EG7 binding. Error bars, S.E. of three independent experiments (p < 0.001). Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S2.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Transfection, Activation Assay, Flow Cytometry, Expressing, Construct, Staining, Software, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-1 is necessary to maintain the co-activator function of K1 in talin-H mediated integrin activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K1 and human K2. B, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused K1, wild type or its mutants, and DsRed talin-H. After 24 h, activation of the integrin was quantified by flow cytometry as in Fig. 1. Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S3.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-3 is dispensable for K3 functions. A, alignment of C-terminal sequences of human K2 and K3; B, αIIbβ3 integrin co-activation analysis using αIIbβ3-CHO cells transfected with EGFP-fused wild-type K3 or K3 chimera and talin-H; the activation of the integrin was determined after 24 h by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S4.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: Extracellular matrix sensing by β1 and β3 integrins controls invadosome formation and localization. (A) Most SrcYF-expressing cells formed invadosome rosettes (visualized by F-actin staining, in green) only on the adhesive surface (gelatin-tetramethylrhodamine B isothiocyanate [TRITC], in red, mixed with vitronectin) and not on antiadhesive areas (Pluronic F127, black areas). (B) Invadosome rosettes stained by phalloidin-TRITC show higher affinity for vitronectin-FITC (light gray bands), sensed by members of the β3 integrin family, than for fibronectin (black bands), sensed by members of both the β1 and β3 integrin families. (C) The β1 and β3 integrins show distinct patterns of localization, observed in MEF-SrcYF cells. β3 (in green in merge) highly colocalized with F-actin, whereas β1 staining is limited to the rosette periphery. (D) 9EG7 antibody β1 staining is specific of the activated form of the integrin. Antibody against activated form of β1 directly conjugated to FITC (9EG7-FITC) was added externally and localized around the invadosome visualized by cortactin-mRFP (in red in merge) expressed in live MEF-SrcYF cells. Bars, 5 μm (A and B), 4 μm (C), and 2 μm (D).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: β1A, and not β3, integrin is essential for invadosome formation. (A) β3 is not essential for invadosome formation and self-assembly into rosettes, visualized by F-actin (in red in merge) and phospho-Y397-FAK (in blue in merge) staining, which occurs in both MEF-SrcYF β3 +/+ or −/− cells. (B) In contrast, β1 depletion in MEF-SrcYF β1 LoxP/LoxP expressing the CRE recombinase for 96 h resulted in the disappearance of isolated invadosomes or in rosettes probed by phalloidin staining. (C) Quantification of the percentage of cells forming invadosomes reveals that almost 95% of MEF-SrcYF β1 LoxP/LoxP treated with CRE recombinase did not form this structure 4 d after infection (n = 650 counted cells/condition). (D) Quantification by qPCR shows an average decrease of 95% in the level of β1 mRNA 96 h post-CRE treatment. Bars, 3 μm (A) and 10 μm (B).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Staining, Expressing, Isolation, Infection

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: Activation of β1 stimulates invadosome autoassembly. (A) β1 depletion in pOBL-SrcYF induced the disappearance of invadosomes, shown by phalloidin staining of F-actin. Bar, 5 μm. (B) β1 depletion does not affect Src activation. Lysates of pOBL-SrcYF β1 +/+ and −/− cells were probed by Western blotting for phospho-SrcY416, a marker of Src activation, total Src, and actin. (C) Amino-acyl sequence of the cytoplasmic domain of β1A integrin and the location of the main mutation that activates this integrin. (D) Expression of the preactivated mutants of β1 (β1 D759A) in pOBL-SrcYF β1 −/− cells dramatically increases the number of invadosome rosettes per airy unit, whereas β1WT mutant rescue the rosette number up to the control level.

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Activation Assay, Staining, Western Blot, Marker, Sequencing, Mutagenesis, Expressing

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: PKC regulates invadosome autoassembly by phosphorylating Ser785 of β1A integrins. (A) PKC activation by a 60-min treatment of pOBL-SrcYF cells with 2 μM PMA induces a massive increase in invadosome rosette autoassembly, visualized by phalloidin staining. (B) Extracted images from time series (in minutes) from representative observations of pOBL-SrcYF cells expressing GFP-actin and treated with either dimethyl sulfoxide (DMSO; control), the PKC activator PMA (2 μM), and the PKC inhibitor BIM (5 mM). PKC activity regulates the dynamics and maintenance of the invadosome autoassembly state. (C) Amino-acyl sequence of the cytoplasmic domain of β1A integrin and the location of the main PKC targets. (D) Quantification of invadosomes per Airy unit (AU) shows that a mutation mimicking a constitutive phosphorylated form of Ser785 (Asp or Glu) dramatically increases the formation of rosettes in pOBL-SrcYF β1 −/− cells. The nonphosphorylatable mutant of β1 at this site (S785A) has no effect on rosette formation. (E) The number of rosettes/AU was quantified in pOBL-SrcYF cells treated with 2 μM PMA or simultaneously with PMA and 5 mM BIM for 60 min. The inhibitory effect of BIM was determined by calculating the percent inhibition of invadosome rosette formation. Mutants mimicking a constitutively phosphorylated Ser785 (S785D and S785E) show only 50% inhibition after BIM treatment, indicating that this residue on β1 is a major target of PKC in the regulation of invadosome autoassembly. Bars, 20 μm (A) and 5 μm (B).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Activation Assay, Staining, Expressing, Activity Assay, Sequencing, Mutagenesis, Inhibition

Journal:

Article Title: Polarized Entry of Uropathogenic Afa/Dr Diffusely Adhering Escherichia coli Strain IH11128 into Human Epithelial Cells: Evidence for ? 5 ? 1 Integrin Recognition and Subsequent Internalization through a Pathway Involving Caveolae and Dynamic Unstable Microtubules

doi: 10.1128/IAI.69.3.1856-1868.2001

Figure Lengend Snippet: Polarized entry of Dr + bacteria into fully differentiated Caco-2/TC7 cells a

Article Snippet: Polyclonal anti-α 5 β 1 integrin was from Biovalley (Conches, France).

Techniques: Infection

Journal:

Article Title: Polarized Entry of Uropathogenic Afa/Dr Diffusely Adhering Escherichia coli Strain IH11128 into Human Epithelial Cells: Evidence for ? 5 ? 1 Integrin Recognition and Subsequent Internalization through a Pathway Involving Caveolae and Dynamic Unstable Microtubules

doi: 10.1128/IAI.69.3.1856-1868.2001

Figure Lengend Snippet: Inhibition of invasiveness of IH11128 bacteria within HeLa cells by polyclonal antibodies directed against ICAM-1, α5β1 integrin, and CD55. Monolayers were incubated with antibodies diluted 1:20 to 1:50 in culture medium for 1 h prior to infection. The cells were infected apically for 3 h at a concentration of 108 CFU/well. Both incubation with antibodies and infection were conducted at 37°C in a 5% CO2–90% air atmosphere. The infected cells were processed for determination of cell-associated and internalized bacteria as described in Materials and Methods. Results represent the means ± standard deviations of three experiments. Student's t test showed a highly significant difference (P < 0.01) for internalized bacteria between control and anti-α5β1 integrin antibody-treated cells. No significant difference was observed for internalized bacteria between control and anti-ICAM1 antibody-treated cells. Note that the highly significant decrease in internalized bacteria in the presence of anti-DAF antibody results from a highly significant decrease in cell association.

Article Snippet: Polyclonal anti-α 5 β 1 integrin was from Biovalley (Conches, France).

Techniques: Inhibition, Incubation, Infection, Concentration Assay

Journal:

Article Title: Polarized Entry of Uropathogenic Afa/Dr Diffusely Adhering Escherichia coli Strain IH11128 into Human Epithelial Cells: Evidence for ? 5 ? 1 Integrin Recognition and Subsequent Internalization through a Pathway Involving Caveolae and Dynamic Unstable Microtubules

doi: 10.1128/IAI.69.3.1856-1868.2001

Figure Lengend Snippet: Entry of laboratory strain E. coli K-12 EC901 carrying the recombinant plasmid pBJN406, which expresses Dr hemagglutinin, within HeLa cells. Experimental conditions were as for experiments shown in Fig. ​Fig.5.5. Results represent the means ± standard deviations of three experiments. Student's t test showed a highly significant difference (P < 0.01) for internalized bacteria between control and anti-α5β1 integrin antibody-treated cells.

Article Snippet: Polyclonal anti-α 5 β 1 integrin was from Biovalley (Conches, France).

Techniques: Recombinant, Plasmid Preparation

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: BMBC demonstrate differential GEF-H1 and PKCα protein expression and HPSE treatment modulated SDC4 CT association. A. GEF-H1 and PKCa are SDC CT associated proteins in HPSE-treated BMBC cells. Whole cell lysates from BMBC cells treated with L- or A-HPSE were immunoblotted for GEF-H1, PKCa, SDC4 or β1Integrin to determine protein expression levels in different BMBC cell lines. Blots were probed for β-actin as a loading control. Data presented are representative of four reproducible experiments. B. The production of GST fusion proteins. Affinity glutathione-S-transferase pulldowns for SDC1 and SDC4 were performed as previously described (11). Figure indicates eluted lysates (1 mg/lane) run on a 15% SDS-PAGE gel under reducing conditions. C. GST pulldown of GEF-H1 by SDC1/4CT. D. GST pulldowns of PKCa by SDC1/4CT. BMBC cells were treated with or without latent or active (L- or A-HPSE) (100ng/ml for 1 hr. at 37°C), then whole cell lysates were generated, passed over GST-SDC1/4 CT fusion protein affinity columns. The same lysates were loaded onto GST protein affinity columns followed by immunoblotting for GEF-H1 or PKCa as controls. Refer to the “Materials and Methods” section for additional details.

Article Snippet: The antibodies and dilutions used in experiments were: SDC1 (1:1,000, clone B-A38), Grb2 (1:1,000, 3972), purchased from Cell Sciences (Canton, MA), SDC4 (1:1,000, ab24511) was from Abcam (Cambridge, MA), full-length PKCα (1:1,000, #2506), GEF-H1 (1:1,000, #4076) and GAPDH (1:1,000, 14C10) were from Cell Signaling (Danvers, MA), β1Integrin (1:1,000, clone 656) was from BD Biosciences (Sparks, MD), Rac1 (1:1,000, clone 23A8) and RhoA (1:500, clone 55) were from Millipore (Danvers, MA), Tiam1 (1:1,000, sc872) and β-actin (1:1,000, sc-69879) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, SDS Page, Generated, Western Blot

Journal: JCI Insight

Article Title: Targetable purinergic receptors P2Y 12 and A2b antagonistically regulate bladder function

doi: 10.1172/jci.insight.122112

Figure Lengend Snippet: Deletion of P2Y12 and A2b result in enlarged (A) and reduced (B) bladder size, respectively. (C–E) H&E staining images from bladder sections of wild-type, P2Y12-KO, and A2b-KO mice, respectively. (F–H) Immunostaining of Ki67 from bladder sections of wild-type (n = 5), P2Y12-KO (n = 5), and A2b-KO (n = 5) mice, respectively. Positive nuclear staining per bladder section is quantitated in I. (J–L) Integrin β1 immunostaining of bladder sections of wild-type (n = 186 cells), P2Y12-KO (n = 225 cells), and A2b-KO (n = 185 cells) mice, respectively. (M–O) Enlarged images of the white boxes seen in J, K, and L, respectively. (P) Schematic diagram indicating where the bladder was sectioned for staining, and how only bladder smooth muscle (BSM) cells containing nuclei were measured for their cross-section area, which serves as an index of BSM cell size. (Q) BSM cell cross-sectional area was quantitated for each model. (R and S) Quantitative RT-PCR data for c-fos and c-jun mRNA expression in mouse bladder from wild-type (n = 3), P2Y12-KO (n = 3), and A2b-KO (n = 3) mice. (T) Western blot images of c-fos and c-jun protein expression in mouse bladder from wild-type (n = 6), P2Y12-KO (n = 6), and A2b-KO (n = 6) mice, which are quantitated by densitometry and normalized to β-actin in U and V. Data are shown as box and whiskers, whiskers are from minimum to maximum, Student’s t test is used to compare between wild-type and KO animals; *P < 0.05.

Article Snippet: Fixed tissue was cryoprotected, frozen, sectioned, and incubated with rabbit polyclonal anti-Ki67 antibody (catalog ab15580, abcam) and purified rat anti-mouse β1 integrin antibody (catalog 550531, BD Bioscience) (1:100 dilution) overnight at 4°C.

Techniques: Staining, Immunostaining, Quantitative RT-PCR, Expressing, Western Blot