integrin β2 myfp  (Addgene inc)

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 1. Integrin β2 can be substituted by its human homolog in mouse Hoxb8 FL cells. (A) Scheme of workflow. Hoxb8 FL cells were generated from bone marrow of integrin β2-deficient mice and retrovirally transduced with mouse or human integrin β2. Human/mouse integrin β2-positive cells were FACS-sorted. (B) Surface expression of different integrin subunits and cell markers on neutrophil-like cells differentiated from control (β2+/+), integrin β2 ko (β2−/−), and human or mouse integrin β2-rescued integrin β2−/−(β2−/−/hβ2 and β2−/−/mβ2) Hoxb8 FL cells assessed by FACS analysis. (C) Integrin β2 surface expression levels on neutrophil-like cells differentiated from integrin β2−/−Hoxb8 FL cells retrovirally transduced with human integrin β2 (β2−/−/hβ2) compared to PMNs isolated from human blood. (D) Static adhesion of untreated, TNFα-treated, or PMA-treated Hoxb8 FL-derived β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 neutrophils on ICAM1. N = 4. Individual data points of the 4 independent experiments are shown. (E,F) Adhesion (E) and rolling velocities (F) of neutrophil-like cells differentiated from β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 Hoxb8 FL cells in flow chambers coated with ICAM1 and P-selectin with or without CXCL1 under constant shear rate of 1 dyn/cm2. N = 10/11 chambers with CXCL1 (rolling and adhesion); N = 3/4 without CXCL1 (adhesion). (G) Neutrophil-like β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 cells plated on a P-selectin-, ICAM1-, and CXCL1-coated surface for 10 min. Scale bar: 100 µm. All values are given as mean ± SD. * p < 0.05, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Generated, Transduction, Expressing, Control, Isolation, Derivative Assay, Shear

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 2. Expression of human integrin β2 rescues spreading and adhesion defects in mouse integrin β2 knockout macrophages. (A) Adhesion of integrin β2−/−and human or mouse integrin β2 ex- pressing integrin β2−/−macrophages to ICAM1 and fibronectin in relation to control macrophages. N = 5. Individual data points represent the 5 independent experiments. (B) Spreading of control, integrin β2−/−, and human or mouse integrin β2 expressing integrin β2−/−macrophages to ICAM1 and fibronectin assessed 2 h after plating. N = 3 independent experiments, shown as individual data points. (C) Hoxb8-derived β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 macrophages plated on an ICAM1- or fibronectin-coated surface for 2 h. Scale bar: 100 µm. (D) Alignment of the amino acid sequence of the mouse and human β2 integrin cytoplasmic tails. Talin-binding NPLF and kindlin- binding NPKF motives are highlighted in red. MP, membrane proximal; MD, membrane distal. (E) Control, integrin β2−/−, and human or mouse integrin β2 expressing integrin β2−/−

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Knock-Out, Control, Derivative Assay, Sequencing, Binding Assay, Membrane

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 3. Expression of human integrin β2 in mouse integrin β2−/−Hoxb8 cells allows assessment of integrin activity using conformation-specific anti-human integrin β2 antibodies. (A,B) FACS analyses of integrin β2 activation of Hoxb8-derived neutrophils expressing human or mouse integrin β2 by measuring staining intensities of conformation-specific antibodies mAb24 (A) and KIM127 (B) either untreated, EDTA-treated, or in response to TNFα, fMLP, or PMA. (C,D) Relative mAb24 (C) and KIM127 (D) binding to resting or EDTA-, TNFα-, fMLP-, or PMA-stimulated Hoxb8-derived neutrophil-like cells upon treatment with DMSO, the phosphatidylinositol-3-kinase (PI3K) inhibitor Wortmannin, the phospholipase C (PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor Gö6983. N = 7 (mAb24) or 5 (KIM127) independent experiments, shown as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Activity Assay, Activation Assay, Derivative Assay, Staining, Binding Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 4. Both talin1 and kindlin3 are required for β2 integrin activation. (A) Scheme of work- flow. Talin1, kindlin3, and talin1/kindlin3 double-deficient Hoxb8 FL cells were generated via the CRISPR/Cas9 system. Single cell clones were screened for talin1 and kindlin3 expression. Four clones per genotype were subjected to CRISPR/Cas9-mediated integrin β2 ablation and retrovirally trans- duced to express human integrin β2. Mouse integrin β2-negative and human integrin β2-positive cells were FACS-sorted. (B) Western blot analysis of neutrophil-like cells differentiated from different human integrin β2 expressing Hoxb8 single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. GAPDH served as loading control. (C,D) Relative mAb24 (C) and KIM127 (D) binding to neutrophil-like cells derived from Hoxb8 single cell clones, expressing human integrin β2 and lacking talin1 and/or kindlin3 expression. N = 4 clones. Result of each clone is plotted as individual data point. All values are given as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Activation Assay, Generated, CRISPR, Clone Assay, Expressing, Western Blot, Control, Binding Assay, Derivative Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 5. Kindlin3 is dispensable for P-selectin-induced integrin αLβ2-mediated slow rolling but required for chemokine-induced slower rolling. (A–C) Adhesion (A), rolling velocities (B), and rolling velocities after LFA-1 blocking (C) of Hoxb8 cell-derived PMN-LCs in flow chambers coated with ICAM1, P-selectin, and CXCL1 under constant shear rate of 1 dyn/cm2. N = 12–16 (A,B) and 6–9 (C) flow chambers. (D) Rolling velocities of PMN-LCs on ICAM1- and P-selectin-coated surfaces (without CXCL1) under constant shear rate of 1 dyn/cm2. N = 8–9 flow chambers. Cells were differentiated from different single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. Rolling velocities are shown as cumulative distribution of the velocities of approximately 500 (B), 300 (C), and 400 (D) cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Blocking Assay, Derivative Assay, Shear, Clone Assay, CRISPR

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 6. Talin and/or kindlin3 binding-deficient human integrin β2 exhibit impaired integrin ac- tivation. (A) Scheme of the integrin β2 cytoplasmic domain, which binds talin1 via a membrane proximal NPLF motif and kindlin3 via a membrane-distal NPKF and a TTT motif (left). Muta- tion of these motifs to NPLA and AAA prevent talin1 and kindlin3 binding, respectively (right). (B) Relative mAb24 binding to neutrophil-like cells derived from Hoxb8 cells expressing human wild-type integrin β2, mutant integrin β2 F/A (F754A), mutant integrin β2 TTT/AAA (T758A, T759A, T760A), or double-mutant integrin F/A TTT/AAA in response to TNFα, CXCL1, fMLP, and PMA or left untreated. N = 5 experiments indicated as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Binding Assay, Membrane, Derivative Assay, Expressing, Mutagenesis

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 7. Rapid CRISPR-based screening approach to study putative β2 integrin regulators. (A) Scheme

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: CRISPR

Journal: Nature cell biology

Article Title: Coupling of β2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis

doi: 10.1038/s41556-019-0414-2

Figure Lengend Snippet: a, Immunofluorescence localization of vinculin, a-actinin, zyxin, phospho-tyrosine, FAK phosphorylated on tyrosine 397 (Y397), paxillin phosphorylated on tyrosine 31 (Y31), syk phosphorylated on tyrosine 342 (Y342) (top panels, and green) in RAW macrophages phagocytosing iC3b-opsonized 5.15 μm polystyrene beads, imaged by confocal microscopy. Actin filaments were labeled with fluorescent phalloidin (middle panels, and red). b, Immunofluorescence of paxillin phosphorylated on tyrosine 118 (Y118, middle right and red) and labeling of actin filaments with fluorescent phalloidin (middle left and green), at the frustrated phagocytic cup of a RAW macrophage on an anti-αMβ2 coated coverslips, imaged by TIRF-SIM. c, Time-lapse TIRF microscopy of the leading edge of a RAW macrophage expressing mEmerald-Paxillin (green) and mCherry-F-tractin (red) during formation of a frustrated phagocytic cup on an anti-αMβ2 coated coverslip. d, Time lapse TIRF microscopy of a RAW macrophage co-expressing αM, β2-EYFP (green) and mApple-Paxillin (red) during formation of a frustrated phagocytic cup on an anti-αMβ2 coated coverslip. Images are representative examples from 3 independent experiments. Scale bars are 5 μm, except b left panel is 2 μm. Elapsed times are in seconds.

Article Snippet: Plasmids for the expression of Mac-1 and integrin β2-EYFP were purchased from Addgene. siRNAs were purchased from Ambion.

Techniques: Immunofluorescence, Confocal Microscopy, Labeling, Microscopy, Expressing

Journal: Nature cell biology

Article Title: Coupling of β2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis

doi: 10.1038/s41556-019-0414-2

Figure Lengend Snippet: a, Fluorescent speckle microscopy during phagocytosis of iC3b-opsonized 5.15 μm polystyrene beads by RAW macrophages expressing Actin-mEos3.2, photoswitched with 405 nm light, and imaged with 561 nm light by time-lapse spinning disk confocal microscopy, from 4 independent experiments. Images were aligned with respect to the ingested particle. Red circles represent detected speckles in the corresponding frame, red lines represent tracks of the speckles from the first time point of their detection to the current frame, as analyzed using automated speckle-tracking software. b, Time lapse TIRF-SIM images of a RAW macrophage expressing F-tractin-mNeonGreen during formation of a frustrated phagocytic cup on anti-αMβ2 coated coverslip, from 3 independent experiments. c, Examples of kymographs generated by line scans perpendicular to the leading edge taken from time-lapse movies during frustrated phagocytosis by F-tractin-EGFP-expressing RAW macrophages spreading on anti-αMβ2-coated coverslips (left) or poly-L-lysine (PLL)-coated coverslips in the presence of 2 mM EDTA to inhibit integrin-ligand engagement (center). Initial leading edge protrusion and actin retrograde flow velocities (lamellipodium= 0-3μm from the leading edge, lamellum= 3-8μm from the leading edge) were quantified from such kymographs (n=5 experiments). Scale bars are 5 μm, except b left panel is 2 μm. Error bars represent SEM, p values are from two tailed Mann-Whitney test. Numerical source data are provided in Statistical Source Data Figure 3.

Article Snippet: Plasmids for the expression of Mac-1 and integrin β2-EYFP were purchased from Addgene. siRNAs were purchased from Ambion.

Techniques: Microscopy, Expressing, Confocal Microscopy, Software, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

doi: 10.1038/srep11630

Figure Lengend Snippet: Dissociation constant (K d ) values determined for integrin β2 tail and Dok1 interactions *.

Article Snippet: Cells (2 × 10 6 ) were transfected with integrin αL plasmid (9 μg), integrin β2-YFP plasmid (WT or mutant) (9 μg) and Dok1-CFP or CFP plasmid (12 μg) by electroporation using the Neon electroporation kit (Invitrogen) on a MP-100 pipette-type microporator (Invitrogen).

Techniques:

Journal: Scientific Reports

Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

doi: 10.1038/srep11630

Figure Lengend Snippet: ( A ) K562 cells transfected with the indicated expression plasmids were lysed. Proteins were resolved on 10% SDS-PAGE under reducing conditions and immunoblotted (IB) with either anti-GFP or anti-Dok1 antibodies. ( B ) Transfected K562 cells were subjected to YFP-photobleach FRET analyses. Each data point represents the mean ± S.D. of ≥30 cells analyzed. ( C ) Flow cytometry analyses of K562 stable line expressing Dok1-CFP that were transfected with integrin αLβ2-YFP and CCR5. Wild-type K562 cells were used as the control group. ( D ) FRET analyses of cells in ( C ) that were treated without or with chemokine RANTES (50 ng/ml) for 10 min at 37 °C. 60 and 39 cells were analyzed for conditions without and with RANTES treatment, respectively. Data point represent mean ± S.D. *,p < 0.05, Student’s t test.

Article Snippet: Cells (2 × 10 6 ) were transfected with integrin αL plasmid (9 μg), integrin β2-YFP plasmid (WT or mutant) (9 μg) and Dok1-CFP or CFP plasmid (12 μg) by electroporation using the Neon electroporation kit (Invitrogen) on a MP-100 pipette-type microporator (Invitrogen).

Techniques: Transfection, Expressing, SDS Page, Flow Cytometry

Journal: Scientific Reports

Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

doi: 10.1038/srep11630

Figure Lengend Snippet: Comparison of amino acid sequences of integrin cytoplasmic tails highlighting the NxxY/F motif (blue) and potential phosphorylation of Ser residues (red).

Article Snippet: Cells (2 × 10 6 ) were transfected with integrin αL plasmid (9 μg), integrin β2-YFP plasmid (WT or mutant) (9 μg) and Dok1-CFP or CFP plasmid (12 μg) by electroporation using the Neon electroporation kit (Invitrogen) on a MP-100 pipette-type microporator (Invitrogen).

Techniques: