Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) Schematic diagram of the potential N -glycosylation sites (N X (S/T)) on the ß4 integrin subunit. The sites corresponding to the putative N -glycosylation sites on the ß4 integrin subunit (Asn 327 , Asn 491 , Asn 579 , Asn 617 , and Asn 695 ) are shown by flags. Numbers and boxes indicate the number of amino acid residue and the four intracellular fibronectin type III repeats, respectively. TM, transmembrane region. (B) Cell lysates from normal keratinocytes were immunoprecipitated using a control IgG or an anti-ß4 integrin Ab. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the biotinylated L 4 -PHA lectin (upper left panel) and E 4 -PHA lectin (upper right panel) or anti-ß4 and anti-α6 integrin Abs (lower panels). IB, immunoblot. The black and white arrowheads indicate the ß4 integrin and α6 integrin subunits, respectively. Ordinates indicate molecular sizes in kDa of marker proteins. (C) Cell morphology of the WT and ΔNß4 integrin-expressing keratinocytes during cell culture. (D) The cell-spreading area in A was calculated using computer software (Image J). Each bar represents the mean ± S.D. of triplicate assays. *p<0.001 (unpaired t-test vs.WT). (E) 20 µg of cell lysates from control lacZ- (lac), WTß4, ΔNß4 integrin-expressing keratinocytes were run on a 6% gel under reducing conditions, blotted onto a nitrocellulose membrane, and then probed with an anti-ß4 integrin Ab. (F) Cell lysates were immunoprecipitated using a polyclonal Ab against ß4 integrin. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the indicated biotinylated lectins or an anti-ß4 integrin Ab. (G) Cell surface expression levels of α3, α6, ß1 and ß4 integrin subunits of WT or ΔNß4 integrin-expressing keratinocytes were examined using FACS analysis. Prior to analysis, cells were incubated with either the indicated integrin Abs or control IgG, followed by incubation with Alexa Fluor conjugated secondary Abs, as described under “ .” (H) Immunoprecipitates from WT or ΔN keratinocytes with an anti-α6 integrin were probed with an anti-ß4 (upper panel) or ß1 integrin (lower panel) Ab.

Article Snippet: A mouse mAb against integrin ß1 (P5D2) and a rat mAb against integrin α5 (BIIG2), which were obtained from Developmental Studies Hybridoma Bank, University of Iowa, were purified from the hybridoma supernatant using a protein A-SepharoseTM 4 Fast Flow column (GE Healthcare).

Techniques: Glycoproteomics, Residue, Immunoprecipitation, Control, Western Blot, Marker, Expressing, Cell Culture, Software, Membrane, Incubation

Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) Cell morphology of the indicated keratinocytes was photographed after cell spreading on laminin-332 substrate for 20 min. (B) The effect of ΔNß4 integrin on cell adhesion activity on 2 µg/ml of the laminin-332 substrate. The experiment was performed in triplicate thrice. *P<0.001 (one-way ANOVA, Bonferroni post test) vs. WT. (C) Effect of ΔNß4 integrin on the cell migration activity on laminin-332 substrate. The indicated keratinocytes in culture medium were plated onto dishes precoated with 2 µg/ml laminin-332, followed by incubation for 1 h. Cell migration was monitored by time-lapse microscopy, as described in “ .” Each bar represents the mean ± S.D. of the migration distance of ten cells in each assay in three independent experiments. *P<0.001 (one way ANOVA, Bonferroni post test) vs. WT. (D) Cell morphology of each keratinocyte during migration. (E) The indicated keratinocytes in culture medium were plated onto dishes precoated with 2 µg/ml laminin-332. After incubation for 24 h, cells were fixed and stained with a paxillin mAb and followed by secondary Ab and phalloidin for visualizing paxillin (green) and actin filament (red). (F) Polarized cells were determined and counted as described in “ .”

Article Snippet: A mouse mAb against integrin ß1 (P5D2) and a rat mAb against integrin α5 (BIIG2), which were obtained from Developmental Studies Hybridoma Bank, University of Iowa, were purified from the hybridoma supernatant using a protein A-SepharoseTM 4 Fast Flow column (GE Healthcare).

Techniques: Activity Assay, Migration, Incubation, Time-lapse Microscopy, Staining

Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) After lysis in 1% Brij98, WT or ΔNß4-expressing keratinocytes were fractionated by sucrose gradient ultracentrifugation. The fractions from each WT and ΔN keratinocytes were independently blotted to the membrane and probed with antibody against ß4 integrin (ß4), and then reprobed with antibody against caveolin-1 (cav-1). (B) Fractions 4 and 5, which were lipid raft fractions, were blotted onto the same membrane and probed with antibody to ß4 integrin (ß4), and then reprobed with antibody to caveolin-1 (cav-1). (C) Results of the densitometric analysis are shown as the integrated density of the ratio of ß4 integrin to caveolin-1 bands in , which was 1.0 for fraction 4 from WT keratinocytes. *P<0.001 (one-way ANOVA, Bonferroni post test) vs. WT. All blots are representative for at least three independent experiments.

Article Snippet: A mouse mAb against integrin ß1 (P5D2) and a rat mAb against integrin α5 (BIIG2), which were obtained from Developmental Studies Hybridoma Bank, University of Iowa, were purified from the hybridoma supernatant using a protein A-SepharoseTM 4 Fast Flow column (GE Healthcare).

Techniques: Lysis, Expressing, Membrane

Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) WT and ΔN keratinocytes were incubated on ice for 30 min in the presence of rat anti-ß4 integrin mAb. The cells were washed and then incubated in the presence (cross-linking, +) or absence (cross-linking, –) of a rabbit anti-rat secondary Ab at 37°C for 10 min. Following incubation, the cells were analyzed for the phosphorylation of ERK by immunoblotting. (B) Results of the densitometric analysis are shown as the integrated density of the ratio of phospho-ERK (pERK) to total ERK (tERK) bands, and the ratio in WT keratinocytes with cross-linking (WT, +) was 1.0. *P>0.05, **p<0.001 (one-way ANOVA, Bonferroni post test) vs. WT with cross-linking. All blots are representative of at least three independent experiments.

Article Snippet: A mouse mAb against integrin ß1 (P5D2) and a rat mAb against integrin α5 (BIIG2), which were obtained from Developmental Studies Hybridoma Bank, University of Iowa, were purified from the hybridoma supernatant using a protein A-SepharoseTM 4 Fast Flow column (GE Healthcare).

Techniques: Incubation, Phospho-proteomics, Western Blot

Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: Under normal conditions (WT ß4 integrin), ß4 integrin binds to EGFR and laminin-332 (Lm332) through cross-linking with galectin-3-mediated N -glycans, which induces a modest association among them, and thereby efficient integrin clustering, cellular signaling, and cell migration. However, in ΔNß4 integrin-expressing keratinocytes, the lack of galectin-3-meditaed ß4 integrin cross-linking and alternation in N -glycosylation of EGFR, resulted in a strong protein-protein association between ß4 integrin and EGFR, and a weak association between ß4 integrin and laminin-332, which inhibited efficient integrin clustering, thereby suppressing cellular signaling and cell migration. The N -glycan of ß4 integrin is also important for the lipid raft localization where SFKs-mediated phosphorylation takes place.

Article Snippet: A mouse mAb against integrin ß1 (P5D2) and a rat mAb against integrin α5 (BIIG2), which were obtained from Developmental Studies Hybridoma Bank, University of Iowa, were purified from the hybridoma supernatant using a protein A-SepharoseTM 4 Fast Flow column (GE Healthcare).

Techniques: Migration, Expressing, Glycoproteomics, Phospho-proteomics

Journal: Frontiers in Physiology

Article Title: Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle

doi: 10.3389/fphys.2022.913673

Figure Lengend Snippet: The role of integrin signaling in agonist-induced constrictor responses with age was assessed in the presence of a functional integrin blocking RGD peptide, a non-blocking RGE peptide, and control no treatment condition. Maximal constrictor responses to NE (10 –4 M), PE (10 –4 M), and Ang II (10 –7 M) in SFA are presented. n = 5–12 rats per group. Data shown are mean ± SE. Significance was evaluated at p < 0.05. *Values are significantly different from young. # Values are significantly different from age-matched control and RGE group.

Article Snippet: The following antibodies were used: rabbit anti-integrin α2 (Abcam, Boston, MA); rabbit anti-integrin α5 (Millipore, Temecula, CA); hamster anti-integrin-β1 conjugated with Alexa 488 and hamster anti-integrin-β3 conjugated with Alexa 488 (Biolegends, San Diego, CA); mouse anti-smooth muscle α-actin (Sigma, St. Louis, MO, United States); goat anti-mouse Alexa 568; goat anti-rabbit Alexa 488 or Alexa 568 (ThermoFischer Scientific, Waltham, MA).

Techniques: Functional Assay, Blocking Assay

Journal: Frontiers in Physiology

Article Title: Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle

doi: 10.3389/fphys.2022.913673

Figure Lengend Snippet: Representative TIRF images of VSM cells isolated from young and old Fischer 344 rats fluorescently labeled for integrin α5, β1 and β3 are shown. Scale bar represents 10 μm. Quantitative measurements ( n = 29–39) are presented as mean ± SE. *Significance was evaluated at p < 0.05.

Article Snippet: The following antibodies were used: rabbit anti-integrin α2 (Abcam, Boston, MA); rabbit anti-integrin α5 (Millipore, Temecula, CA); hamster anti-integrin-β1 conjugated with Alexa 488 and hamster anti-integrin-β3 conjugated with Alexa 488 (Biolegends, San Diego, CA); mouse anti-smooth muscle α-actin (Sigma, St. Louis, MO, United States); goat anti-mouse Alexa 568; goat anti-rabbit Alexa 488 or Alexa 568 (ThermoFischer Scientific, Waltham, MA).

Techniques: Isolation, Labeling

Journal: Frontiers in Physiology

Article Title: Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle

doi: 10.3389/fphys.2022.913673

Figure Lengend Snippet: (A) Representative TIRF images of VSM cells plated on fibronectin and fluorescently labeled for integrin α5, β1 and β3 are shown. (B) Representative TIRF images of VSM cells plated on collagen-I and fluorescently labeled for integrin α2 (red) and β1 (green) are shown. Scale bar represents 10 μm. Quantitative measurements ( n = 15–50) are presented as mean ± SE. Significance was evaluated at p < 0.05.

Article Snippet: The following antibodies were used: rabbit anti-integrin α2 (Abcam, Boston, MA); rabbit anti-integrin α5 (Millipore, Temecula, CA); hamster anti-integrin-β1 conjugated with Alexa 488 and hamster anti-integrin-β3 conjugated with Alexa 488 (Biolegends, San Diego, CA); mouse anti-smooth muscle α-actin (Sigma, St. Louis, MO, United States); goat anti-mouse Alexa 568; goat anti-rabbit Alexa 488 or Alexa 568 (ThermoFischer Scientific, Waltham, MA).

Techniques: Labeling

Journal: Frontiers in Physiology

Article Title: Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle

doi: 10.3389/fphys.2022.913673

Figure Lengend Snippet: (A) Adhesion assay of VSM cells to fibronectin was performed in the presence of a functional integrin blocking RGD peptide and a non-blocking RGE peptide. Negative control experiments were performed using untreated cells plated on substrates blocked with BSA, and specific matrix without cells. Data are presented as mean ± SEM. (B) Functional AFM measurements of integrin α5β1 binding force to fibronectin for young and old VSM cells treated with a functional integrin blocking RGD peptide and a non-blocking RGE peptide are shown. For comparisons, force peak values whose confidence intervals did not overlap were considered significantly different ( p < 0.05). (C) Percent of adhesion events for each treatment is shown.

Article Snippet: The following antibodies were used: rabbit anti-integrin α2 (Abcam, Boston, MA); rabbit anti-integrin α5 (Millipore, Temecula, CA); hamster anti-integrin-β1 conjugated with Alexa 488 and hamster anti-integrin-β3 conjugated with Alexa 488 (Biolegends, San Diego, CA); mouse anti-smooth muscle α-actin (Sigma, St. Louis, MO, United States); goat anti-mouse Alexa 568; goat anti-rabbit Alexa 488 or Alexa 568 (ThermoFischer Scientific, Waltham, MA).

Techniques: Cell Adhesion Assay, Functional Assay, Blocking Assay, Negative Control, Binding Assay

Journal: Frontiers in Physiology

Article Title: Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle

doi: 10.3389/fphys.2022.913673

Figure Lengend Snippet: Representative confocal images of VSM cells plated on (A) rigid and (B) soft substrates functionalized with fibronectin and collagen-I, respectively, are shown. VSM cells have been fluorescently labeled for integrin β1. Scale bar represents 10 μm. Quantitative measurements of overall fluorescence intensity ( n = 28–68) are presented as mean ± SE. Significance was evaluated at p < 0.05. *Values are significantly different from young. # Values are significantly different from age matched VSM cells on different matrices. $ Values are significantly different between matrices.

Article Snippet: The following antibodies were used: rabbit anti-integrin α2 (Abcam, Boston, MA); rabbit anti-integrin α5 (Millipore, Temecula, CA); hamster anti-integrin-β1 conjugated with Alexa 488 and hamster anti-integrin-β3 conjugated with Alexa 488 (Biolegends, San Diego, CA); mouse anti-smooth muscle α-actin (Sigma, St. Louis, MO, United States); goat anti-mouse Alexa 568; goat anti-rabbit Alexa 488 or Alexa 568 (ThermoFischer Scientific, Waltham, MA).

Techniques: Labeling, Fluorescence

Journal: Frontiers in Physiology

Article Title: Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle

doi: 10.3389/fphys.2022.913673

Figure Lengend Snippet: Representative confocal images of VSM cells plated on soft substrates functionalized with fibronectin (A) and collagen-I (B) are shown. VSM cells have been fluorescently labeled for integrin β1. Scale bar represents 10 μm. Quantification of fluorescence intensity relative to inner vs. outer cell area shows a strong recruitment of integrin β1 at cell edges for old cells plated on fibronectin ( n = 28–30), with a modest relative change for cells plated on collagen-I ( n = 40–50). Data are presented as mean ± SE. Significance was evaluated at p < 0.05. *Values are significantly different from young. #Values are significantly different from age matched VSM cells.

Article Snippet: The following antibodies were used: rabbit anti-integrin α2 (Abcam, Boston, MA); rabbit anti-integrin α5 (Millipore, Temecula, CA); hamster anti-integrin-β1 conjugated with Alexa 488 and hamster anti-integrin-β3 conjugated with Alexa 488 (Biolegends, San Diego, CA); mouse anti-smooth muscle α-actin (Sigma, St. Louis, MO, United States); goat anti-mouse Alexa 568; goat anti-rabbit Alexa 488 or Alexa 568 (ThermoFischer Scientific, Waltham, MA).

Techniques: Labeling, Fluorescence