Journal: iScience

Article Title: Noradrenergic pathway from the locus coeruleus to heart is implicated in modulating SUDEP

doi: 10.1016/j.isci.2023.106284

Figure Lengend Snippet:

Article Snippet: For β1-AR staining, sections were incubated at 4°C overnight in a solution of rabbit anti-β1-AR (1:400; AAR-023, Alomone), followed by incubation in a solution of secondary antibodies (donkey anti-rabbit Alexa 546, 1:1000; A10040, Thermo Fisher Scientific) for 2 h at room temperature.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: Conserved residues in the C-terminal region of kindlin-2 support αIIbβ3 activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K2 and Fit1, the K2 homolog of D. melanogaster. B and C, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused FL, wild-type K2 or its mutants and DsRed talin-H, and activation of αIIbβ3 integrin was determined at 24 h by flow cytometry by staining the cells with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S1.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A, HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars, S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B, HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars, S.D. (p < 0.001). C, mouse RAW 264.7 cells were transiently transfected with plasmids encoding EGFP alone or EGFP-K2 constructs. The transfected cells were stained with 9EG7 monoclonal antibody to assess β1 integrin activation. Flow cytometry was used to measure 9EG7 binding. Error bars, S.E. of three independent experiments (p < 0.001). Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S2.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Transfection, Activation Assay, Flow Cytometry, Expressing, Construct, Staining, Software, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-1 is necessary to maintain the co-activator function of K1 in talin-H mediated integrin activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K1 and human K2. B, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused K1, wild type or its mutants, and DsRed talin-H. After 24 h, activation of the integrin was quantified by flow cytometry as in Fig. 1. Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S3.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-3 is dispensable for K3 functions. A, alignment of C-terminal sequences of human K2 and K3; B, αIIbβ3 integrin co-activation analysis using αIIbβ3-CHO cells transfected with EGFP-fused wild-type K3 or K3 chimera and talin-H; the activation of the integrin was determined after 24 h by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S4.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry