Journal: Cell Host & Microbe

Article Title: β1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections

doi: 10.1016/j.chom.2017.05.001

Figure Lengend Snippet:

Article Snippet: The samples were stained with a rat anti-mouse β1-integrin (1:100 dilution, clone MB1.2, Merck Millipore), anti-acid ceramidase (1:100 dilution), anti-ceramide (1:100 dilution), anti-sphingosine (1:1000 dilution), anti-mouse β2-integrin antibodies (1:100, clone M1812, 1:100, #557437, BD) or FITC-Annexin (1:200, #11 828 681 001, Roche) in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl 2 , 0.7 mM MgCl 2 , 0.8 mM MgSO 4 ) plus 1% FCS at room temperature for 45 min.

Techniques: Virus, Isolation, Recombinant, Software

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) Domain architecture of an active (open headpiece/extended) integrin α5β1 heterodimer. α5 Subunit is grey; β1 subunit headpiece and leg are, respectively, in shades of blue and green. The localization of three different mAb epitopes, exposed only in the conformationally active β1 subunit, is represented. Epitopes of mAb 12G10 and mAb HUTS4, respectively, lie in the βI domain and hybrid domain of the headpiece, whereas mAb 9EG7 epitope is in the I-EGF2 domain. (B) Confocal immunofluorescence microscopy analysis of the subcellular localization of the three different anti-active β1 integrin mAbs employed to stain fixed ECs. All three mAbs bind to active β1 integrins mainly located within typical elongated fibrillar adhesions. Scale bar 20 µ m; magnification scale bar 10 µ m. (C) Confocal immunofluorescence microscopy analysis of anti-active β1 integrin mAbs localization after 10 min of incubation on living ECs. Anti-I-EGF2 domain mAb 9EG7 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, whereas anti-βI domain mAb 12G10 recognizes active β1 integrins located both outside and inside highly fragmented and tiny adhesions. Similar to mAb 9EG7, the anti-hybrid domain mAb HUTS4 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, hinting that anti-active headpiece mAb-elicited fragmentation specifically depends on βI domain binding. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Selected frames from (top row) and (bottom rows), respectively, illustrating dynamic mAb 9EG7-Alexa Fluor 488 binding to active β1 integrins over time upon live incubation on ECs either in the absence (top row) or in the presence (bottom rows) of mAb 12G10–Alexa Fluor 647. When incubated alone (top row), mAb 9EG7–Alexa Fluor 488 preferentially binds active β1 integrins located within fibrillar adhesions and remains stable over time. When mAb 12G10–Alexa Fluor 647 is pre-incubated on ECs, mAb 9EG7–Alexa Fluor 488 does no longer localize in fibrillar adhesions. Scale bar 20 µ m. (E) Representative g -STED confocal microscopy pictures of anti-active β1 integrin 9EG7 mAb localization after 10-min incubation on living ECs either in the absence (top left panel) or the presence (middle left panel) of 12G10 or TS2/16 (bottom left panel). To thoroughly analyze the morphology of ECM adhesion sites, g -STED confocal images were acquired close to the basal EC surface. 9EG7-labeled adhesions were then analyzed with ImageJ software (right panels) and classified, according to their shape factor (SF), into elongated (red) and round (yellow) structures. 9EG7-labeled adhesions were classified as elongated, if their SF was < 0.5, and round, if the SF was ≥ 0.5. Scale bar 20 µ m. The maximum Feret’s diameter was measured to quantify the morphological features of 9EG7 + elongated structures. Compared with control ECs incubated live with 9EG7 alone, 9EG7 + elongated structures were significantly shortened in the presence of 12G10 or TS2/16. Data are mean ± SD, n ≥ 20 cells per condition pooled from two independent experiments. 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, Staining, Incubation, Binding Assay, Confocal Microscopy, Labeling, Software, Control

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: Low magnification confocal immunofluorescence microscopy analysis of ECs that were treated live for 10 min with either mAb 9EG7 mAb ( green ) alone or in combination with mAb 12G10 ( red ). Scale bar 100 µ m.

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

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

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) Representative g -STED confocal microscopy analysis of tensin 1 localization in ECs that were incubated or not for 10 min with the anti-active β1 integrin mAb 9EG7 or mAb 12G10 or the Fab fragment of mAb 12G10 (12G10-Fab) or mAb TS2/16. Scale bar 20 µ m; magnification scale bar 10 µ m. When compared with untreated control (CTL) ECs or those treated with mAb 9EG7, the mFD of tensin 1 + fibrillar adhesions was significantly reduced in ECs treated with either mAb 12G10, 12G10-Fab or mAb TS2/16. Data are mean ± SD, n ≥ 22 cells per condition pooled from three independent experiments. The number of structures was normalized on cell area and on those in control cells. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (B) MAb 9EG7 affects the lifetime of FN–α5β1 bonds. AFM measurement of mAb 9EG7 effect on force-dependent lifetime of single bonds between a FNIII 7–10 fragment and an integrin α5β1-Fc fusion protein. Lifetime versus force plots of α5β1-Fc–functionalized Petri dish dissociating from FNIII 7–10 -coated cantilever tips in Mn 2+ either in the absence (grey) or the presence (green) of 10 µ g/ml mAb 9EG7 mAb. Data are mean ± SEM of several tens to several hundreds of measurements per point. 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

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) Selected frames from (A), (B), and (C), monitoring mAb 12G10 ( red ) binding to active β1 integrins on living: control ECs (A); ECs previously oligofected with tensin-EGFP (B); ECs pre-incubated for 10 min with mAb 9EG7 (C). (A, B, C) As expected, mAb 12G10 mAb binds active β1 integrins located within and outside fragmented adhesion sites (A), but, either when Tensin-EGFP is overexpressed (B) or upon pre-incubation with mAb 9EG7 (C), mAb 12G10 mAb localizes instead within fibrillar adhesions that remain stable over time. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Representative confocal images showing pre-bleaching, post-bleaching, and recovery on the region of interest (indicated by arrows) of tensin-EGFP–positive fibrillar adhesions in ECs treated with DMSO (as control) or with the FAK inhibitor PF-562271. Scale bar 5 µ m. Recovery rate was measured, and data were normalized by employing as reference the fluorescence intensity acquired on the same ROI before bleaching. Data were then normalized on control DMSO-treated samples. Data are mean ± SD, n ≥ 31 adhesions per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.001 ***. The effectiveness of inhibition of FAK autophosphorylation by PF-562271 was verified by Western blot analysis of EC lysates. (E) Representative confocal microscopy images of anti-active β1 mAb 12G10 ( green ) localization in ECs treated or not with the FAK inhibitor PF-562271; ECs were also stained for auto-phosphorylated FAK on tyrosine 397 (pFAK-Y397, red ). Scale bar 20 µ m; magnification scale bar 10 µ m. Measurement of mFD of 12G10 + adhesions revealed that, compared with control EC incubated with DMSO, 12G10 + adhesive structures are significantly longer in presence of FAK inhibitor PF-562271. Data are mean ± SD, n ≥ 19 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (F) Western blot analysis of FAK autophosphorylation (Y397) levels in ECs treated with or without mAb 9EG7 or mAb 12G10 for 2 or 15 min. 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: Binding Assay, Control, Incubation, Fluorescence, Inhibition, Western Blot, Confocal Microscopy, Staining, Adhesive

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: Representative confocal microscopy analysis of the impact of mAb 12G10 on living ECs previously incubated for 10 min either with intact mAb 9EG7–Alexa Fluor 488 (9EG7) or its Fab (9EG7-Fab). Scale bar 10 µ m; magnification scale bar 10 µ m. Upon pre-incubation with intact dimeric 9EG7, but not monomeric 9EG7-Fab, mAb 12G10 localizes within fibrillar adhesions. Quantitative analysis showed that the incubation of cultured ECs with 12G10 shortens the mFD of elongated active β1 integrin + clusters only when cells are pre-incubated with monomeric 9EG7-Fab, but not intact dimeric 9EG7. Data are mean ± S.D, n ≥ 22 cells per condition pooled from three independent experiments. Statistical analysis: one-way ANOVA, 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: Confocal Microscopy, Incubation, Cell Culture

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) Confocal immunofluorescence microscopy analysis of ECs live treated with mAb 9EG7 ( green ) alone or in combination with mAb 12G10 ( blue ). The internalization of mAb 9EG7–bound active β1 integrins in EEA1 + early endosomes ( red ) was quantified by Pearson correlation coefficient (PCC). Treatment with the anti-βI domain mAb 12G10 promotes mAb 9EG7–bound active β1 integrin endocytosis. Data are mean ± SD, n ≥ 23 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 ****. (B) Time-course analysis of the relative amounts of endocytosed 9EG7 + active β1 integrins in control (CTL; green ) versus mAb 12G10–treated ( light blue ) ECs, evaluated by internalization and capture ELISA assays. Treating living ECs with the anti-βI domain mAb 12G10 elicits a strong increase in 9EG7 + active β1 integrin endocytosis. Data are mean ± SD, of eight technical replicates per condition pooled from three independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (C, D) Real-time analysis of EC haptotactic migration towards FN (xCELLigence RTCA DP system) either in the absence (CTL) or the presence of anti-active β1 integrin mAb 9EG7 alone (C, D) or mAb12G10 alone (C) or combined mAb 9EG7 and mAb 12G10 (D). Data are mean ± SD, n ≥ 14 technical replicates per condition pooled from four independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.05 * , # ; P ≤ 0.01 ** , ## ; P ≤ 0.001 *** , ### ; P ≤ 0.0001 **** , #### . (C, D) *CTL versus mAb 9EG7; # CTL versus mAb 12G10 (C); or CTL versus mAb 9EG7 + mAb 12G10 (D). 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, Control, Enzyme-linked Immunosorbent Assay, Migration

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

Journal: PLoS Biology

Article Title: BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGFβ responses and altered cell mechanics

doi: 10.1371/journal.pbio.3000557

Figure Lengend Snippet: (A) Immunoblot showing total β1-integrin (tITGB1) levels and phosphorylation at Ser785 and Threonine 783 (left) under steady-state conditions and densitometric quantification of total or phosphorylated β1-integrin levels in indicated cell clones (right). Data are presented as mean + SD ( n = 3–4 independent experiments). (B) PLA between active β1-integrin (ITGB1 pS785 ) and ILK under steady-state growth conditions. (C) Single confocal z-planes of BMPR2 wt (basal, left), BMPR2 ΔE2 (medial, middle), and BMPR2 KO (medial, right) depicting the localization of ILK (green) or paxillin (red). Insets show zoomed-in regions. (D) Immunoblots showing protein levels of VE-Cadherin and phosphorylated VE-Cadherin in indicated ECs under steady-state conditions. (E) Single confocal z-planes of immunocytochemical staining using antibodies specific for VE-Cadherin (green) and PECAM-1 (red). Insets show zoomed-in regions. (F) Immunoblots showing levels of phosphorylated cofilin (pSer-3), pMLC (pSer-19), and total MLC in indicated ECs under steady-state culture conditions. (G) Cartoon (left) depicting the principle of CFS (particle diameter 23 μm). Elastic modulus derived from CFS of living cells (middle). Representative indentation (force versus distance) curves for the different cell lines and the hard control surface (mica; right). Data are shown as mean + SD ( n = 30). (H) Principle of QI using a sharp cantilever tip (diameter < 20 nm; left). Representative QI scans of fixed cells, focusing on CCC sites (middle). Height profiles of nucleus-to-cell junction height differences (black arrows) are indicated. Given values are expressed as the mean ± SD. Height profile measurements were taken from n ≥ 18 cells; scale bars, 10 μm; Statistical significance relative to BMPR2 wt was calculated using Kruskal-Wallis test with post hoc Dunn test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also and for underlying data. BMP, bone morphogenetic protein; BMPR2, BMP type-2 receptor; CCC, cell-to-cell contact; CFS, colloidal force spectroscopy; EC, endothelial cell; ILK, integrin-linked kinase; MLC, myosin light chain; PECAM-1, platelet endothelial cell adhesion molecule-1; PLA, proximity ligation assay; pMLC, phosphorylated MLC; QI, quantitative imaging; pS785, phosphorylated Serine 785; pT783, phosphorylated Threonine 783; tITGB1, total integrin beta-1; VE, vascular endothelial.

Article Snippet: Respective dilutions are indicated as follows: phosphorylated SMAD1/5, clone 41D10 (WB 1:1000; Cell Signaling Technologies); phosphorylated SMAD2, clone 138D4 (WB 1:1000; Cell Signaling Technologies); SMAD1, clone D59D7 (WB 1:1000; Cell Signaling Technologies, ChIP 5 μg/immunoprecipitation); SMAD1, AB55476 (PLA 1:200; Abcam); SMAD2 clone 86F7 (WB 1:1000; PLA 1:200; Cell Signaling Technologies); SMAD2/3 clone 18 (WB 1:1500; PLA 1:200; BD Biosciences); SMAD5 12167-1-AP (WB 1:1000; PLA 1:100; Proteintech); SMAD4 clone D3R4N (WB 1:1000; PLA 1:800; Cell Signaling Technologies); ILK clone 65–1 (WB 1:500; ICC 1:50; PLA 1:50; Santa Cruz Biotechnology); β1-integrin (CD-29) clone 18 (WB 1:1000; BD Biosciences); β1-integrin clone P5D2 (ICC 1:500; Abcam); phosphorylated β1-integrin-Ser785 AB8124 (WB 1:10000; PLA 1:500; Merck Millipore); phosphorylated β1-integrin-Tyr783 AB8125 (WB 1:10000; Merck Millipore); phosphorylated paxillin-Tyr118 (ICC 1:100; Cell Signaling Technologies); N-Cadherin clone 32 (ICC 1:250; BD Biosciences); VE-Cadherin clone D87F2 (WB 1:1000); phosphorylated VE-Cadherin-Tyr685 CP1981 (WB 1:1000; ECM Biosciences); VE-Cadherin clone BV6 (ICC 1:200; Merck Millipore); PECAM-1 (CD-31) clone 89C2 (ICC 1:3000; immunohistochemistry [IHC]: 1:250; Cell Signaling Technologies); LTBP-1 (IHC: 1:1000; the antibody was previously described [ ]); β-Catenin clone D10A8 (ICC 1:250; Cell Signaling Technologies); αSMA (IHC: 1:250 ab5694, Abcam); MLC 2 #3672 (WB 1:1000; Cell Signaling Technologies); pMLC-Ser19 #3671 (WB 1:1000; ICC 1:50; IHC: 1:50; Cell Signaling Technologies); phosphorylated cofilin-Ser3 clone 77G2 (WB 1:1000; Cell Signaling Technologies); LAP (of TGFβ-1) clone 9005 (WB 1: 1000; ICC 1:250; R&D Systems); TβR2 clone C-4 (WB: 1:500; Santa Cruz Biotechnology); BMPR2 (ICC 1:200; Cell Signaling Technologies); BMPR2 clone-18 (WB 1:1000; BD Biosciences); FN ab23750 (ICC 1:50; Abcam); GAPDH clone 14C10 (WB 1:1000; Cell Signaling Technologies); β-Actin clone AC-15 (WB 1:5000; Sigma Aldrich); myc-tag clone 9B11 (WB 1:1000; ICC 1:8000; Cell Signaling Technologies); and TGFβ-1,2,3 MAB1835 (WB 1:1000; R&D Systems).

Techniques: Western Blot, Clone Assay, Staining, Derivative Assay, Spectroscopy, Proximity Ligation Assay, Imaging

Journal: The Journal of Cell Biology

Article Title: Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

doi: 10.1083/jcb.200404076

Figure Lengend Snippet: Fyn is required for a laminin-mediated switch in NRG signaling and for integrin activation to increase survival. (A) Survival of newly formed SFK-depleted oligodendrocytes in the presence of the PI3K pathway signaling inhibitor wortmannin (hatched bars), the MAPK pathway signaling inhibitor PD098059 (light gray bars), or DMSO control (black bars). Lm2 switches NRG-mediated survival from PI3K-sensitive to PI3K-insensitive, and Fyn depletion, but not Lyn depletion, abolishes this effect. Error bars represent SD. (B) Oligodendrocytes treated for 30 min with NRG. PhosphoERK is enhanced by Lm2 in control cells, but not in Fyn(−) cells. (C) Survival of newly formed SFK-depleted oligodendrocytes in the presence (light gray bars) or absence (dark gray bars) of integrin-activating manganese. Integrin activation using manganese increased NRG-mediated survival, and this increase was lost in the absence of Fyn, but not of other SFKs. Error bars represent SD.

Article Snippet: The following mouse monoclonals were used: GFP; Fyn, and Csk (Transduction); pp60src (Oncogene Research Products); a rat mAb against MBP (Serotec); a hamster IgM against β1 integrin (BD Biosciences); and FITC or TRITC donkey secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Activation Assay

Journal: The Journal of Cell Biology

Article Title: Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

doi: 10.1083/jcb.200404076

Figure Lengend Snippet: SFK associations with integrins and growth factors. (A) Newly formed oligodendrocytes immunostained with antibodies against Fyn (red) and α6β1 integrin (green). Merged panel is shown on the right. (B) Oligodendrocyte lysates from cells differentiated on ECM substrates in the presence or absence of PDGF or NRG. Western blot on α6 integrin antibody immunoprecipitation complexes to detect Fyn. (C) Western blot on Fyn antibody immunoprecipitation complexes to detect ErbB4 NRG receptor subunit. (D) Western blot on PDGFαR antibody immunoprecipitation complexes to detect Lyn. (E) Western blots on Lyn antibody immunoprecipitation complexes to detect the β3 integrin subunit.

Article Snippet: The following mouse monoclonals were used: GFP; Fyn, and Csk (Transduction); pp60src (Oncogene Research Products); a rat mAb against MBP (Serotec); a hamster IgM against β1 integrin (BD Biosciences); and FITC or TRITC donkey secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

doi: 10.1083/jcb.200404076

Figure Lengend Snippet: SFK activity is regulated by laminin in differentiating oligodendrocytes, not progenitors. (A) Active Lyn is detected in response to the αVβ3 ligand FN. Immunoprecipitation complexes using antibodies against Fyn, Lyn, or control mouse IgG (−) were evaluated by Western blot for the presence of autophosphorylated SFK phosphoY418. (B) Expression and solubility of Fyn, Lyn, and COOH-terminal Src kinase (Csk) change during oligodendrocyte lineage progression and in response to Lm2. Western blots were performed using antibodies specific for SFKs and SFK regulatory kinase Csk. S, Triton X-100–soluble protein; I, Triton X-100–insoluble protein. Blots were also probed with actin antibodies as protein loading controls. (C) Phosphorylation of the SFK negative regulatory site is reduced by laminin in oligodendrocytes, not progenitors. The same lysates as in B were used, but Western blots were analyzed using antibodies against two SFK sites: phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). (D) FN does not alter phosphorylation of the SFK negative regulatory site. Lysates of progenitors and oligodendrocytes grown on control substrate PDL or on the αVβ3 ligand FN. Western blots were performed using SFK antibodies specific for phosphoY418 (catalytic) and phosphoY527 (COOH-terminal negative regulatory). Blots were also probed with actin antibodies as protein loading controls. (A–D) In the absence of ECM ligands, cells were grown on non-integrin substrate PDL.

Article Snippet: The following mouse monoclonals were used: GFP; Fyn, and Csk (Transduction); pp60src (Oncogene Research Products); a rat mAb against MBP (Serotec); a hamster IgM against β1 integrin (BD Biosciences); and FITC or TRITC donkey secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Expressing, Solubility

Journal: The Journal of Cell Biology

Article Title: Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

doi: 10.1083/jcb.200404076

Figure Lengend Snippet: Model for integrin regulation of SFK activity during oligodendrocyte lineage progression. In oligodendrocyte progenitors, Lyn is associated with the PDGFαR–αVβ3 integrin complex and contributes to proliferation signaling. Catalytic Y397 of Lyn (orange) is phosphorylated after αVβ3 integrin ligation. Fyn is maintained in the inactive state by Csk phosphorylation of Fyn-inhibitory COOH-terminal Y531 (yellow). After axonal contact and ligation by α6β1 of the α2 chain laminins expressed in myelinating axon tracts, Lyn dissociates from the integrin–growth factor complex and Csk is downregulated, reducing Fyn phosphorylation at Y531 and promoting Fyn activity. Active Fyn–α6β1 complexes can then trigger PI3K and MAPK signaling, depending on the ligand binding of PDGFαR and ErbB2/4 receptors, respectively, thereby promoting oligodendrocyte survival, differentiation, and myelin formation.

Article Snippet: The following mouse monoclonals were used: GFP; Fyn, and Csk (Transduction); pp60src (Oncogene Research Products); a rat mAb against MBP (Serotec); a hamster IgM against β1 integrin (BD Biosciences); and FITC or TRITC donkey secondary antibodies (Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, Ligation, Ligand Binding Assay

Journal:

Article Title: Cardiac dysfunction in aging conscious rats: altered cardiac cytoskeletal proteins as a potential mechanism

doi: 10.1152/ajpheart.00146.2008

Figure Lengend Snippet: Comparison of expression levels of β1-integrin, vinculin, α-actinin, and talin in young and old LV rat samples. There were no significant differences in the expression of these proteins.

Article Snippet: The following antibodies were used: β 1 -integrin, 610468 (Becton Dickinson); α-tubulin, T6199 (Sigma); β-tubulin, T7816 (Sigma); desmin, D8281(Sigma); α-actinin, A5044 (Sigma); vinculin, V9133 (Sigma); and talin, T3287 (Sigma).

Techniques: Comparison, Expressing