Journal: Cell reports

Article Title: PAI-1 uncouples integrin-β1 from restrain by membrane-bound β-catenin to promote collagen fibril remodeling in obesity-related neoplasms

doi: 10.1016/j.celrep.2024.114527

Figure Lengend Snippet:

Article Snippet: For DKK1-, SPARC-, TIMP2-, COL1, integrin β2, or integrin α3 PLA, purified rabbit monoclonal anti-DKK1 (1:200, Cell Signaling Technology, Cat# 48367S), rabbit polyclonal anti-SPARC (1:200, Cell Signaling Technology, Cat# 5420S), mouse monoclonal anti-TIMP2 (1:200, R&D Systems, Cat# MAB971), mouse monoclonal anti-COL1 (1:200, Cell Signaling Technology, Cat# 66948S), goat polyclonal anti-integrin β1 (1:200, R&D Systems, Cat# AF1778), goat polyclonal anti-integrin β2 (1:200, R&D Systems, Cat# AF1730), and mouse monoclonal anti-integrin α3 (1:200, R&D Systems, Cat# MAB1345) were used.

Techniques: Recombinant, Membrane, Cell Culture, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Protein Extraction, Extraction, Bicinchoninic Acid Protein Assay, In Situ, Blocking Assay, Migration, shRNA, Control, Software, Pyromark Assay, Western Blot, Simple Western

Journal: The Journal of biological chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites.

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 5. Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B, targeting of TMEM2 to focal adhesions (FAs) does not require the cyto- plasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 (full length) and mCherry-mTMEM2/Δcyto (Δcyto) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A, in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in Experimental procedures section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B, immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C–E, TMEM2 associates with integrins via extracellular interactions. C, cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3’,3’-dithiobis(sulfosuccinimidyl

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane

Journal: The Journal of biological chemistry

Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites.

doi: 10.1016/j.jbc.2021.100481

Figure Lengend Snippet: Figure 6. A model for the role of transmembrane protein 2 (TMEM2) in integrin-mediated cell adhesion and migration. Our results suggest that TMEM2-dependent degradation of hyaluronan (HA) is critical for cells to form strong cell–matrix adhesion on HA-rich extracellular matrix (ECM). A, high levels of HA in the ECM are inhibitory to the direct engagement of integrins to their ECM ligands. B, in the presence of TMEM2, HA in the ECM is locally removed, which generates a microenvironment that is permissible to the direct integrin–ECM engagement. C, the association between TMEM2 and integrins promotes the FA formation and maturation via further removal of HA in the vicinity of the integrin–ECM engagement. D, this in turn facilitates integrin clustering, integrin-mediated downstream signaling, and cellular responses. See the text for further discussion.

Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS++ overnight at 4 C. After extensive washing, bound materials were eluted by boiling in SDS-PAGE sample buffer, and eluents were analyzed by SDSPAGE and immunoblotting with rabbit polyclonal antiintegrin α5 (Proteintech; 10569-1-AP), rabbit monoclonal anti-integrin β1 (Abcam; ab52971), rabbit polyclonal antiintegrin β2 (Proteintech; 10544-1-AP), or mouse monoclonal anti-polyhistidine (Sigma; A7058; clone: HIS-1, peroxidase conjugated).

Techniques: Migration

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Primer sequences and PCR settings

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Hybridization

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Expression of CD11a and CD18 in melanoma A375, 1205LU and SLM8 cell lines with conditioned medium. Cell-surface expression of CD11a and CD18 on indicated melanoma cell lines treated for 24 hrs with HUVEC conditioned medium was analyzed by flow cytometry. Isotypic controls are represented as empty histograms and specific antibody-labelling is displayed as shaded histograms. Histograms obtained with cells incubated with FCS-complete medium and labeled with specific antibodies, which overlap with the isotypic control are not shown. Data from obtained with 3 independent experiments.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Expressing, Flow Cytometry, Incubation, Labeling, Control

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Effect of CD11a and CD18-blocking antibodies on the transendothelial migration of A375, 1205LU and SLM8 cell lines. The experiments were performed as detailed in Figure , except that 2μg/ml of CD11a or CD18-blocking antibodies were introduced in the upper chamber of the Transwells when indicated. Histograms represent 3 independent experiments. In each experiment each condition was analyzed in duplicate.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Blocking Assay, Migration

Journal: BMC Cancer

Article Title: LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

doi: 10.1186/1471-2407-12-455

Figure Lengend Snippet: Effect of CD11 and CD18-blocking antibodies on the formation of clumps. A Semi-quantitative PCRs were performed to detect the expression of the ICAM-1 transcript. GAPDH is used as a DNA amount control. B A375, 1205LU and SLM8 cell lines were treated with 2 μg/ml of CD11a or CD18-blocking antibodies as indicated. Melanoma cells were labeled with DiO then fixed and labeled with DAPI prior to their observation under an epifluorescence microscope using a magnification of x10. Data were obtained from 3 independent experiments.

Article Snippet: Specific direct primary antibodies CD11a antibody (FAB35951A) and CD18 (FAB1730P) from R&D system (Minneapolis, MN, USA) or isotypic control antibody (BD Pharmingen, San Diego, CA, USA) were used at 1 μg ml.

Techniques: Blocking Assay, Expressing, Control, Labeling, Microscopy

Journal: Biochemistry and Biophysics Reports

Article Title: Irisin supports integrin-mediated cell adhesion of lymphocytes

doi: 10.1016/j.bbrep.2021.100977

Figure Lengend Snippet: Immunofluorescent cytometry analysis of integrin expression. Representative FACS histograms of flow cytometry analysis illustrating the expression of different integrin subunits on (A) Jurkat cells, (B) PBMCs, and (C) TK1 cells. The background staining with isotype control antibodies is shown as black lines, whereas specific monoclonal antibody staining is shown as red lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Briefly, recombinant human integrin α4β7 (Cat: 5397-A3, R&D Systems), recombinant human integrin αLβ2 (Cat: 3868-AV, R&D Systems), or recombinant mouse integrin αLβ2 (Cat: 7825-AB, R&D Systems) to the concentration of 3 μg/ml was immobilized to wells of a 96-well transparent microtiter plates and incubated overnight at 4 °C.

Techniques: Cytometry, Expressing, Flow Cytometry, Staining, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Irisin supports integrin-mediated cell adhesion of lymphocytes

doi: 10.1016/j.bbrep.2021.100977

Figure Lengend Snippet: Purified protein based-ELISA type experiments to study the interaction of irisin with integrin αLβ2 and α4β7. Binding of irisin- or control-Fc to immobilized proteins such as; mouse αLβ2 (A), human αLβ2 (B), human α4β7 (C), and BSA as a control (A–C); was examined. (A–C) Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control Fc in each group.

Article Snippet: Briefly, recombinant human integrin α4β7 (Cat: 5397-A3, R&D Systems), recombinant human integrin αLβ2 (Cat: 3868-AV, R&D Systems), or recombinant mouse integrin αLβ2 (Cat: 7825-AB, R&D Systems) to the concentration of 3 μg/ml was immobilized to wells of a 96-well transparent microtiter plates and incubated overnight at 4 °C.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Binding Assay, Control