kindlin 2 merck millipore mab2617 wb  (Proteintech)

Journal: JCI Insight

Article Title: Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation

doi: 10.1172/jci.insight.152559

Figure Lengend Snippet: ( A and B ) Representative immunofluorescence images of dorsal skin sections and fluorescence analysis of DCIR staining in the skin tissues of patients with AD and controls ( n = 8). Scale bar: 100 μm. ( C ) Quantification analysis of DCIR + tryptase + cells in the lesion skin of patients with AD and controls. ( D ) Flow cytometry analysis of DCIR expression in human mast cell line cKit + FcεRI + LAD2 cells. ( E ) Scheme of experimental protocol for the direct bindings of human recombinant DCIR (hrDCIR) to BSA, CRE, and Man-BSA. ( F ) Direct binding of different doses of hrDCIR (0–5.0 μg/mL) to BSA, CRE, or Man-BSA ( n = 3). ( G ) Representative immunofluorescence images of FITC-CRE uptake by LAD2. Scale bar: 15 μm. ( H and I ) Flow cytometry analysis ( H ) and quantification ( I ) of FITC-CRE uptake at different doses (1–500 ng/mL) by LAD2 cells ( n = 3). ( J ) Inhibition of FITC-CRE uptake in LAD2 cells pretreated with DCIR neutralizing antibody (α-DCIR) or IgG isotype ( n = 4). Data represent mean ± SEM of 2 independent experiments. Data in B , C , and I were compared using a 2-tailed Student’s t test. Data in F and J were compared by 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were then incubated with the primary antibodies against mouse Tryptase (AF1937, R&D system), DCIR (MAB2617, R&D), or mouse IgG1 overnight at 4°C.

Techniques: Immunofluorescence, Fluorescence, Staining, Flow Cytometry, Expressing, Recombinant, Binding Assay, Inhibition

Journal: JCI Insight

Article Title: Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation

doi: 10.1172/jci.insight.152559

Figure Lengend Snippet: ( A ) Representative skin images and EASI scores of PBS- and CRE-treated WT and DCIR –/– mice. ( B ) Representative H&E staining and epidermal thickness (μm) of skin tissues of PBS- and CRE-treated WT and DCIR –/– mice. ( C ) Representative Toluidine blue staining and quantification of cells with positive staining for Toluidine blue of skin tissue sections of PBS- and CRE-treated WT and DCIR –/– mice. Scale bar: 100 μm. Arrows represent mast cells. ( D ) Serum levels of specific IgE and IgG1 to CRE. ( E ) Quantitative PCR analysis of IL-4, IL-13, IL-33, and TNF-α expression in the skin tissues of PBS- and CRE-treated WT and DCIR –/– mice. Each circle represents 1 mouse. n = 8. Data represent mean ± SEM of 2 independent experiments. Data were compared by 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were then incubated with the primary antibodies against mouse Tryptase (AF1937, R&D system), DCIR (MAB2617, R&D), or mouse IgG1 overnight at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: JCI Insight

Article Title: Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation

doi: 10.1172/jci.insight.152559

Figure Lengend Snippet: ( A ) Scheme of experimental protocol of i.v. transfer of DCIR + versus DCIR – mast cells into Kit W-sh/W-sh mice for the generation of AD mouse model. ( B ) Representative Toluidine blue staining and quantification of cells with positive staining for Toluidine blue of skin tissue sections of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells ( n = 9). ( C ) Representative immunofluorescence images of mast cells with (yellow) or without (blue) DCIR expression. ( D ) Representative skin images and EASI scores of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells ( n = 8). ( E ) Representative H&E staining and epidermal thickness (μm) of skin tissues of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells ( n = 8). Scale bar: 100 μm. Arrows represent mast cells. ( F ) Serum levels of specific IgE and IgG1 to CRE ( n = 8). ( G ) Quantitative PCR analysis of IL-4, IL-13, IL-33, and TNF-α expression in the skin tissues of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells. Each circle represents 1 mouse ( n = 8). Data represent mean ± SEM of 2 independent experiments. Data were compared by 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were then incubated with the primary antibodies against mouse Tryptase (AF1937, R&D system), DCIR (MAB2617, R&D), or mouse IgG1 overnight at 4°C.

Techniques: Staining, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction

Journal: Nature Structural & Molecular Biology

Article Title: Talin and kindlin use integrin tail allostery and direct binding to activate integrins

doi: 10.1038/s41594-023-01139-9

Figure Lengend Snippet: Reagents used in the study

Article Snippet: Anti-KIND2 (1:1,000) , Merck Millipore, MAB2617.

Techniques: Control, DNA Ligation, Labeling, Chromatography, Purification, Cloning

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: Kindlin-2 is strongly expressed in endothelial cells. A, B HUVECs were treated by VEGFA (50 ng/ml) or not for 24 h and cell extracts were prepared. Representative Western blotting results show increased protein level of KINDLIN-2 in VEGFA-stimulated HUVECs (N = 5 independent experiments, mean ± SD is shown. Student’ t test, **p < 0.01 vs control). C QRT-PCR results show increased mRNA level of KINDLIN-2 in VEGFA-stimulated HUVECs (N = 6 independent experiments, mean ± SD is shown. Student’ t test, **p < 0.01 vs control). D, E HUVECs were treated by TNFα (50 ng/ml) or not for 48 h and cell extracts were prepared. Representative Western blotting results show reduced protein level of KINDLIN-2 in TNFα-stimulated HUVECs (N = 5 independent experiments, mean ± SD is shown. Student’ t test, ****p < 0.0001 vs control). F qRT-PCR results show reduced mRNA level of KINDLIN-2 in TNFα-stimulated HUVECs (N = 5 independent experiments, mean ± SD is shown. Student’ t test, ****p < 0.0001 vs control). G Co-localization of CD31 and KINDLIN-2 was detected in the kidney vasculature of rats. Scale bars: 200 μm

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: Kindlin-2 specific deletion in endothelial cells causes embryonic lethality of mice. A Tie2-cre; Kindlin-2fl/fl (cKO) and Kindlin-2fl/fl (Ctrl) embryos collected at E9.5, E10.5 and E11.5. N = 8 embryos in each group. Scale bars: 2 mm. B Vasculature in Tie2-cre; Kindlin-2fl/fl (cKO) and Kindlin-2fl/fl (Ctrl) embryos at E10.5. N = 8 embryos in each group. Scale bars: 200 μm. C IHC staining of E10.5 embryos with CD31, sections shown are the head and tail parts of embryos. N = 4 embryos in each group. Scale bars: 100 μm. D Immunofluorescent staining of E10.5 embryos with CD31 (green) and Kindlin-2 (red). Sections shown are the back part of embryos. N = 4 embryos in each group. Scale bars: 500 μm and 100 μm

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Immunohistochemistry, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: Impaired angiogenic functions caused by endothelial KINDLIN-2 inhibition is reversed by treatment of NOTCH inhibitor DAPT. A–C Western blotting of NICD, HEY1. HUVECs were transfected with siK2 or siNC for 48 h and treated with or without DAPT (N = 5 independent experiments, mean ± SD is shown. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs siNC. Two-way ANOVA Bonferroni multiple comparison test, ####p < 0.0001). D, E qRT-PCR of HEY1 and HES1. HUVECs were transfected with siK2 or siNC for 48 h and treated with or without DAPT (N = 5 independent experiments, mean ± SD is shown. ****p < 0.001 vs siNC. Two-way ANOVA Bonferroni multiple comparison test, ####p < 0.0001). F–H Immunofluorescent staining of NICD and HEY1. Cells transfected with siK2 or siNC for 48 h and treated with or without DAPT (N = 8 independent experiments. mean ± SD is shown. *p < 0.05, ***p < 0.001, vs siNC. Two-way ANOVA Bonferroni multiple comparison test, #p < 0.05, ####p < 0.0001). Scale bars: 50 μm. I Tube formation of HUVECs, cells were transfected with siK2 or siNC for 48 h and treated with or without DAPT. N = 4 independent experiments. Scale bars:1 μM. J CD31 stained retinal wholemounts from P6 mice. Intravitreally injected with the siNC, siK2 or DAPT at P4. N = 4 mice in each group. Scale bars: 1 mm. K Working model of Kindlin-2 limiting Notch1 signaling. During angiogenesis, endothelial Notch1 is cleaved by γ-secretase. Free Notch1 Intracellular Domains (NICD) are transferred to nucleus to direct the transcription of Hey1 and Hes1. Kindlin-2 interacts with NICD and prevents the cleavage of Notch1. In contrast, Kindlin-2 deficiency facilitates Notch1 signal

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Inhibition, Western Blot, Transfection, Comparison, Quantitative RT-PCR, Staining, Injection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: Kindlin-2 deficiency impairs endothelial basement membrane degradation, cells sprouting, migration, and tube formation. A, B Representative images of gelatin degradation of HUVECs transfected with siNC and siK2 (N = 6 independent experiments, mean ± SD is shown. ***p < 0.001, vs siNC. Two-way ANOVA Bonferroni multiple comparison test, #p < 0.05). Scale bars: 500 μm. C Higher magnification images show ECs sprouting into matrigel and filopodias on activated sprouts. Scale bars: 1 μm. Statistics of number (D) and length (E) of ECs sprouts (N = 6 independent experiments, mean ± SD is shown. Student’ t test, ***p < 0.001 vs siNC). Statistics of filopodias assembled on sprouts (F) (N = 6 independent experiments, mean ± SD is shown. Student’ t test, ***p < 0.001 vs siNC). G Representative phase-contrast images taken at 0 h and 24 h after scraping the confluent monolayer cells showed the wound width and their statistics (H) (N = 8 independent experiments, mean ± SD is shown. *p < 0.05, vs siNC. Two-way ANOVA Bonferroni multiple comparison test, #p < 0.05). Scale bars: 1 mm. I Representative images of tube formation. N = 5 independent experiments. Scale bars: 1 mm. J Western blotting results of KINDLIN-2 in HUVECs transfected by siK2 or siNC. N = 5 independent experiments

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Membrane, Migration, Transfection, Comparison, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: Endothelial KINDLIN-2 controls angiogenic competence via NOTCH1 signaling. A, B Western blotting results of NICD, HEY1, HES1 and NOTCH1 in KINDLIN-2 siRNA transfection group (siK2) or control group (siNC) (N = 5 independent experiments, mean ± SD is shown. Student’ t test, **p < 0.01, ***p < 0.001 vs siNC). C qRT-PCR of NICD, HEY1 and HES1 (N = 5 independent experiments, mean ± SD is shown. Student’ t test, *p < 0.05, **p < 0.01, ***p < 0.001 vs siNC). D–F Immunofluorescence staining and fluorescence intensity of NICD and HEY1 (N = 8 independent experiments, mean ± SD is shown. Student’ t test, **p < 0.01, ***p < 0.001 vs siNC). Scale bars: 50 μm. G, H Proliferation reflected by EdU positive cells (N = 5 independent experiments, mean ± SD is shown. Student’ t test, ***p < 0.001, vs siNC). Scale bars: 200 μm. I–K Immunofluorescence of NICD, Hey1 or Hes1 (red) co-staining with CD31 (green) in mouse embryos of Tie2-cre; Kindlin-2fl/fl (cKO) and Kindlin-2fl/fl (Ctrl). N = 5 independent experiments. Scale bars: 200 μm

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: KINDLIN-2 sustains the NOTCH1 stability by interacting with NICD and preventing the cleavage of NOTCH1. A Immunofluorescence staining of NOTCH1 or NICD (red) and KINDLIN-2 (green) of HUVECs. N = 6 independent experiments. Scale bars: 10 μm. B, C Co-IP assays of V5-NICD and Flag-KINDLIN-2 in HEK293T cells. N = 5 independent experiments. D, E co-IP assays of NICD and KINDLIN-2 in HUVECs. N = 5 independent experiments. F, G MG132 assays of HUVECs transfected by siNC and siK2. The cells were pre-treated by MG132 (10 μg) for 6 h before harvested. N = 5 independent experiments. H, I CHX (100 μg/ml) experiments of HUVECs transfected with siK2 or siNC, samples were harvested at various time points. Protein level of NICD was examined by Western Blotting. N = 5 independent experiments. J, K DAPT treatment of HUVECs transfected with siK2 or siNC, samples were harvested at different time points. Protein level of NICD was detected by Western Blotting. N = 5 independent experiments. L CO-IP assays of NICD and full-length or truncated KINDLIN-2, HUVECS were transfected with full-length or truncated KINDLIN-2 for 48 h, and cell extracts were harvested for CO-IP assays. N = 5 independent experiments

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Transfection, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Kindlin-2 controls angiogenesis through modulating Notch1 signaling

doi: 10.1007/s00018-023-04866-w

Figure Lengend Snippet: KINDLIN-2 deficiency attenuates the capacity of excessive angiogenesis of HUVECs caused by high glucose (HG). A, B Western blotting of KINDLIN-2 (N = 6 independent experiments, mean ± SD is shown. Student’ t test, ***p < 0.001 vs normal glucose). C, D Immunofluorescence staining of KINDLIN-2 (N = 6 independent experiments, mean ± SD is shown. Student’ t test, ***p < 0.001, vs normal glucose). Scale bars: 100 μm. E, F The gelatin degradation assays of HUVECs (N = 5 independent experiments, mean ± SD is shown. ***p < 0.001, ****p < 0.0001 vs siNC. Two-way ANOVA Bonferroni multiple comparison test, ####p < 0.0001). Scale bars: 500 μm. G, H Representative phase-contrast images of HUVECs taken at 0 h and 24 h after scraping the confluent monolayer cells showed the wound width (N = 6 independent experiments, mean ± SD is shown. *p < 0.05, ***p < 0.001, vs siNC. Two-way ANOVA Bonferroni multiple comparison test, #p < 0.05). Scale bars: 1 mM. I Tube formation ability of HUVECs. N = 5 independent experiments. Scale bars: 1 mm. J Higher magnification images of ECs sprouting into matrigel. Statistics of number (K) and length (L) of ECs sprouts (N = 5 independent experiments, mean ± SD is shown, ****p < 0.0001 vs siNC. Two-way ANOVA Bonferroni multiple comparison test, ###p < 0.001). Scale bars: 500 μm. M, N Higher magnification images of filopodias on activated sprouts. (N = 6 independent experiments, mean ± SD is shown. ***p < 0.001, ****p < 0.0001 vs siNC. Two-way ANOVA Bonferroni multiple comparison test, ###p < 0.001). Scale bars: 1 μm

Article Snippet: The antibody against Kindlin-2 (MAB2617) is from Millipore (Billerica, MA, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Comparison