Journal: The Journal of Experimental Medicine

Article Title: Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin

doi: 10.1084/jem.20150718

Figure Lengend Snippet: AKB-9778–triggered stimulation of Tie-2 stabilizes EC contacts via activation of Rap1. (A) HUVECs were treated for 30 min with AKB-9778, COMP-Ang1, or vehicle (as indicated), followed by analyzing the activation level of Rac1 by a luminescence-based G-LISA Rac1 activation assay. (B) HUVECs were either pretreated with solvent (vehicle) or with AKB-9778 for 30 min before solvent (−) or thrombin (+) was added and incubated for 2 min (as indicated), followed by immunoblotting cell lysates for phospho-Thr18/Ser19 of nonmuscle MLC (pMLC2), MLC2, and VE-cadherin. (C) Confluent HUVEC monolayers were treated with either solvent (vehicle) or AKB-9778 for 30 min before thrombin was added and incubated (as indicated) for 15 min, followed by staining of fixed and permeabilized cells for F-actin and VE-cadherin. Bars, 20 µm. (D) HUVECs were either left untreated (−) or treated with AKB-9778 or with COMP-Ang1, followed by a pull-down assay for Rap1 and immunoblotting for this GTPase. (E) HUVECs were transfected with control siRNA or Rap1 siRNA. 72 h later, cells were treated with 10 µM AKB-9778 for 10 min, and Rac1 activity was measured by a pull-down experiment with the effector protein PAK, and active Rac1 was compared with total levels of Rac1. Total levels of Rap1 are shown at the bottom. (F) HUVECs were silenced for Rap1 as in E, grown on transwell filters, and treated for 30 min with thrombin either in the presence of solvent (vehicle) or of AKB-9778 (as indicated). Permeability for 250-kD FITC-dextran was determined as for . (G) Total cell lysates of HUVECs transfected with control siRNA or Rap1-targeting siRNA were immunoblotted for Rap1 and α-tubulin (as indicated). Data are pooled from three independent experiments with three replicates in each experiment (A) or at least two independent experiments (B–G). Statistical significance was analyzed using the one-way ANOVA test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Results are shown as means ± SEM.

Article Snippet: For GST pull-downs of Rac1 or Rap1, cells were serum starved for 2–4 h before stimulation with vehicle, AKB-9778, or COMP-Ang1 for 30 min, followed by lysis in 50 mM Tris/HCl, pH 7.5, 1% Triton X-100, 100 mM NaCl, 10 mM MgCl 2 , 5% glycerol, and protease inhibitors for 10 min. Lysates were centrifuged at 14,000 rpm and 4°C for 10 min and incubated with either Rac1 Assay Reagent (PAK-PBD-GST Beads; Cytoskeleton, Inc.) for a pull-down of Rac1 or Rap1 Assay Reagent (Ral-GDS-RBD Beads; Merck Millipore) for a pull-down of Rap1 for 45 min at 4°C in an overhead shaker.

Techniques: Activation Assay, Solvent, Incubation, Western Blot, Staining, Pull Down Assay, Transfection, Control, Activity Assay, Permeability

Journal: The Journal of Experimental Medicine

Article Title: Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin

doi: 10.1084/jem.20150718

Figure Lengend Snippet: AKB-9778 counteracts LPS-induced leakiness and neutrophil recruitment in the lung even in the absence of VE-cadherin. (A) Cdh5 lox/lox or Cdh5 iECKO mice were i.p. injected with tamoxifen daily for 5 d and, 2 d later, subcutaneously injected with solvent (ctrl or LPS group) or with AKB-9778 and then exposed to nebulized LPS or saline (ctrl) for 40 min. 4 h later, mice were i.v. injected with Evans blue and 5 min later sacrificed; the lung circulation was perfused, and the dye was extracted from the lung tissue and quantified. (B) Mice were subcutaneously injected with either vehicle alone or AKB-9778 and 30 min later i.v. injected with either 60 µg/mouse control IgG or BV13 (as indicated). 2 h later, Evans blue was i.v. injected. After 15 min, mice were sacrificed, the lung circulation was perfused, and the dye was extracted from the lung tissue and quantified. (C) Total lung lysates of mice from D were immunoblotted for the indicated antigens. (D) Cdh5 lox/lox or Cdh5 iECKO mice were treated with tamoxifen and LPS as in A, and extravasated neutrophils were isolated by bronchoalveolar lavage and counted. (E and F) ECs were isolated from the lungs of Cdh5 iECKO mice, treated in culture for 54 h with solvent (EtOH) or 4-hydroxytamoxifen (Tam), and stimulated for 30 min either with vehicle or AKB-9778 (as indicated), followed by a pull-down assay for Rap1 (E) or Rac1 (F) and immunoblotting for the analyzed GTPase. Total cell lysates were immunoblotted for VE-cadherin and α-tubulin (as indicated). Data are pooled from two independent experiments with four mice per group in each experiment (A) or two to three mice per group in each experiment (B) or five mice per group in each experiment (D) and representative of at least two independent experiments (C, E, and F). Statistical significance was analyzed using the one-way ANOVA test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Results are shown as means ± SEM.

Article Snippet: For GST pull-downs of Rac1 or Rap1, cells were serum starved for 2–4 h before stimulation with vehicle, AKB-9778, or COMP-Ang1 for 30 min, followed by lysis in 50 mM Tris/HCl, pH 7.5, 1% Triton X-100, 100 mM NaCl, 10 mM MgCl 2 , 5% glycerol, and protease inhibitors for 10 min. Lysates were centrifuged at 14,000 rpm and 4°C for 10 min and incubated with either Rac1 Assay Reagent (PAK-PBD-GST Beads; Cytoskeleton, Inc.) for a pull-down of Rac1 or Rap1 Assay Reagent (Ral-GDS-RBD Beads; Merck Millipore) for a pull-down of Rap1 for 45 min at 4°C in an overhead shaker.

Techniques: Injection, Solvent, Saline, Control, Isolation, Pull Down Assay, Western Blot

Journal: Frontiers in Medicine

Article Title: Third dose of anti-SARS-CoV-2 inactivated vaccine for patients with RA: Focusing on immunogenicity and effects of RA drugs

doi: 10.3389/fmed.2022.978272

Figure Lengend Snippet: Anti-SARS-CoV-2 NAbs responses from RA patients and HC. (A) NAb titers in RA patients ( n = 222) and HC ( n = 177). Symbols show individual values, and the red line shows the maximum value, and the black horizontal bar shows the median. Statistical analysis was performed using the Mann-Whitney U test. (B) Seropositive rates in RA patients and HC. Statistical analysis was performed using the χ 2 test. (C) Effects of immunomodulatory drugs on NAb titers. RA patients were divided into four groups based on the immunomodulatory drug they were using, including csDMARD ( n = 76), bsDMAD ( n = 24), JAK inhibitors ( n = 24) and prednisone ( n = 46). (D) Effects of TCM on NAb titers. Based on whether they were taking TCM, each RA group mentioned in Figure 2C was further divided into two groups including TCM and no TCM. (E) NAb titers in RA patients with anti-keratin antibody positive or negative compared with HC. (F) Comparison of NAb titers in groups having different interval time. * p < 0.05; ** p < 0.01.

Article Snippet: Serum samples were collected, and the anti-SARS-CoV-2 NAb quantitative detection kit (Spike RBD) (RAS-N044, ACROBiosystems, Beijing, China) was used to measure the NAbs titers.

Techniques: MANN-WHITNEY, Comparison

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1

doi: 10.1681/ASN.2016040477

Figure Lengend Snippet: MR and Rac1 genetic deletion in SMCs prevent oxidative injury induced by IR. (A) Deficiencies of MR and Rac1 in SMCs prevented lipid peroxidation induced by IR. (B) MR deletion in ECs did not prevent lipid peroxidation induced by IR. MDA levels were quantified in kidney lysates. The data were normalized against the protein concentration of the tissue lysate. n=6 per group. Two-way ANOVA was performed. **P<0.001; ***P<0.001.

Article Snippet: Specific antibodies against Rac1 (610650; BD Biosciences, San Jose, CA, and ARC03; Cytoskeleton) and MR (kindly provided by Dr. Gomez-Sanchez; 1:200) were used.

Techniques: Protein Concentration

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1

doi: 10.1681/ASN.2016040477

Figure Lengend Snippet: A lack of Rac1 in SMCs protects against kidney IR injury. Plasma levels of (A) creatinine and (B) urea were measured as an index of renal function. (C) The percentage of injured tubules was quantified blind on ten fields per mouse. Representative images for the hematoxylin and eosin staining are shown for (D) Rac1f/f sham, (E) Rac1f/f IR, (F) Rac1SMCKO sham, and (G) Rac1SMCKO IR. Scale bar, 20 μm. White bars represent the sham groups and black bars represent the IR groups. n=5 per group. Mann–Whitney U test analysis was performed. *P<0.01; **P<0.001; ***P<0.001.

Article Snippet: Specific antibodies against Rac1 (610650; BD Biosciences, San Jose, CA, and ARC03; Cytoskeleton) and MR (kindly provided by Dr. Gomez-Sanchez; 1:200) were used.

Techniques: Clinical Proteomics, Staining, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1

doi: 10.1681/ASN.2016040477

Figure Lengend Snippet: MR activation increases Rac-1 activity and affects oxidative stress. (A) Rac1 activity was determined in rat primary SMCs incubated with aldosterone (Aldo), aldosterone and spironolactone (MRA), or EGF as a positive control. A representative Western blot is shown, together with a densitometric analysis. (B) Hydrogen peroxide production was quantified in rat primary SMCs in the presence of aldosterone, aldosterone and spironolactone, and aldosterone and Rac1 inhibitor (EHT 1864). n=5 per group. One-way ANOVA analysis was performed. *P<0.01 versus control; #P<0.01 versus Aldo; **P<0.01 versus vehicle; †††P<0.01 versus Aldo.

Article Snippet: Specific antibodies against Rac1 (610650; BD Biosciences, San Jose, CA, and ARC03; Cytoskeleton) and MR (kindly provided by Dr. Gomez-Sanchez; 1:200) were used.

Techniques: Activation Assay, Activity Assay, Incubation, Positive Control, Western Blot, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1

doi: 10.1681/ASN.2016040477

Figure Lengend Snippet: MR and Rac1 deficiency in SMCs prevent an inactivating sulfenic acid modification on ETB receptor. The ETB receptor was immunoprecipitated from whole kidneys and a Western blot against cysteine sulfenic acid was performed in (A) MRendoKO mice, (B) MRSMCKO mice, and (C) Rac1SMCKO mice. n=5 per group. Two-way ANOVA was performed. *P<0.01; **P<0.001; ***P<0.001.

Article Snippet: Specific antibodies against Rac1 (610650; BD Biosciences, San Jose, CA, and ARC03; Cytoskeleton) and MR (kindly provided by Dr. Gomez-Sanchez; 1:200) were used.

Techniques: Modification, Immunoprecipitation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1

doi: 10.1681/ASN.2016040477

Figure Lengend Snippet: Proposed mechanism for the benefits of MRAs in renal ischemia. During renal ischemia, MR is activated, signaling to Rac1 and increasing ROS production in the SMCs. The ROS that diffuse to ECs induce a post-translational sulfenic acid modification in endothelin-B receptor (ETBR). When this modification is present, ETBR cannot activate eNOS and therefore NO production is decreased. The decrease in NO generation induces sustained vasoconstriction and reduced kidney perfusion, leading to tubular injury because of decreased oxygen delivery to the cells.

Article Snippet: Specific antibodies against Rac1 (610650; BD Biosciences, San Jose, CA, and ARC03; Cytoskeleton) and MR (kindly provided by Dr. Gomez-Sanchez; 1:200) were used.

Techniques: Modification