mouse anti-rac2  (Abnova)

Journal: The Journal of Experimental Medicine

Article Title: Chemokine-coupled β 2 integrin–induced macrophage Rac2–Myosin IIA interaction regulates VEGF-A mRNA stability and arteriogenesis

doi: 10.1084/jem.20132130

Figure Lengend Snippet: Chemokine-coupled β 2 integrin engagement drives Rac2 activation (GTP-loading), Rac2-dependent HuR translocation, and consequent VEGF mRNA stabilization. (A) Rac2 and Myh9 immunoblots from lysates of WT BMDMs adhered to immobilized ICAM-1 or Fcγ fragment control for 5 min at 37°C in the presence or absence of 50 ng/ml CCL2, after which GTP-Rac2 was affinity precipitated by PBD pulldown ( n = 4). (B) Rac2, Myh9, Actin, and CCR2 immunoblots from lysates of Rac2 +/+ , Rac2 +/− , and Rac2 −/− BMDMs ( n = 3). (C) Relative CCR2 surface expression on thioglycollate-elicited peritoneal macrophages (CD11b hi F4/80 hi ) from Rac2 +/+ (red) and Rac2 −/− (blue) mice (isotype control; black, n = 4). (D) Confocal micrographs of Rac2 +/+ and Rac2 −/− BMDMs, CCL2-stimulated and ICAM-bound for 30 min, followed by HuR immunostaining for translocation analysis. Bar, 10 µm. (E) Quantification of percentage of cells in D with HuR nuclear-to-cytosolic translocation (**, P < 0.002; n = 3). (F) VEGF-A mRNA decay assays, using the indicated BMDMs, adhered to immobilized ICAM-1 or Fcγ fragment control in the presence or absence of CCL2 for 30 min, after which transcription was arrested with 10 mg/ml actinomycin d at time 0, and RNA collected at the noted time points for VEGF-A mRNA quantification by RT-qPCR (**, P = 0.0001; n = 5). Thioglycollate-elicited peritoneal macrophage data are from two independent experiments, each with two mice per treatment group. Remaining data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Article Snippet: Anti-Rac2 mouse monoclonal antibody was purchased from Proteintech.

Techniques: Activation Assay, Translocation Assay, Western Blot, Control, Expressing, Immunostaining, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Chemokine-coupled β 2 integrin–induced macrophage Rac2–Myosin IIA interaction regulates VEGF-A mRNA stability and arteriogenesis

doi: 10.1084/jem.20132130

Figure Lengend Snippet: Chemokine-coupled integrin-driven activation of Rac2 leads to a unique Rac2-Myh9 complex that correlates with HuR translocation, and in vivo neovascular responses depend on efficient integrin activation of Rac2. (A) THP-1 cells in the presence or absence of CCL2 (50 ng/ml × 10 min) were lysed and GTPγS (or vehicle control)-loaded, followed by PBD affinity pulldown and Coomassie staining (top) or immunoblotting for the indicated Rho family GTPases (bottom; n = 3). (B) Chemokine stimulation and GTP loading as in A, in Rac2 +/+ or Rac2 −/− BMDMs, followed by PBD affinity pulldown and Coomassie staining for the 226 kD protein noted above ( n = 3). (C) Immunoblots from lysates of Rac2 +/+ and Rac2 −/− BMDMs, which were CCL2-treated for 10 min, GTPγS (100 µM)-loaded for 10 min, and affinity precipitated with PBD beads to determine the association of Myh9 with the indicated Rac proteins ( n = 4). (D) Confocal micrographs of human peripheral blood monocytes adhered to ICAM-1 or Fcγ fragment control in the presence or absence of 50 ng/ml CCL2 for 10 min, followed by PLA reaction for Rac2–Myh9 interaction (red signal) and DAPI (cyan) staining ( n = 3). Bar, 15 µm. (E) PLA-HuR translocation correlation graph. THP-1 HuR-YFP stable transfectants were ICAM-1– or poly- l -lysine control-bound in the presence or absence of CCL2, after which PLA and HuR translocation analysis were performed ( r = 0.9, P < 0.0001, n = 3). (F) Rac2 and Myh9 immunoblots from lysates of Rac2 +/+ or Rac2 +/− BMDMs adhered to immobilized ICAM-1 in the presence of 50 ng/ml CCL2, after which Rac2 was affinity precipitated by PBD pulldown ( n = 3). (G and H) Laser Doppler scan images (G) of flow in the ischemic (I) and contralateral control (C) hind limb of Rac2 +/+ and Rac2 +/− mice at the indicated time points before and after femoral artery ligation with quantitative blood flow analysis (H; **, P < 0.0025; n = 10). (I) Quantification of CD11b hi F4/80 hi macrophages isolated from collagenase-treated ischemic muscle tissue at day 3 after femoral artery ligation (**, P < 0.0001; n = 6). Animal data are from 2 independent experiments, each with 3 mice per treatment group. Remaining data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Article Snippet: Anti-Rac2 mouse monoclonal antibody was purchased from Proteintech.

Techniques: Activation Assay, Translocation Assay, In Vivo, Control, Staining, Western Blot, Ligation, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Chemokine-coupled β 2 integrin–induced macrophage Rac2–Myosin IIA interaction regulates VEGF-A mRNA stability and arteriogenesis

doi: 10.1084/jem.20132130

Figure Lengend Snippet: Macrophage HuR translocation with consequent VEGF-A mRNA stabilization, flow recovery during hind limb ischemia, and tissue VEGF-A mRNA levels depend on myeloid Myh9. (A) Myh9, Actin, Rac2, and CCR2 immunoblots of lysates obtained from BMDMs isolated from LysM +/cre Myh9 +/+ ( Myh9 +/+ ), LysM +/cre Myh9 +/fl ( Myh9 +/− ), and LysM +/cre Myh9 fl/fl ( Myh9 −/− ) mice ( n = 3). (B) Flow cytometric CD11b and Ly6C phenotyping (top) of density gradient–enriched peripheral blood mononuclear cells obtained from myeloid cell–specific Myh9 +/− and Myh9 −/− mice along with quantification of absolute cell count per ml blood (lower panel, n = 4). (C) Flow cytometric CD11b and F4/80 phenotyping (top) of cells recovered from the peritoneal cavity 3 d after initiation of thioglycollate-induced peritonitis in myeloid cell–specific Myh9 +/− and Myh9 −/− mice along with quantification of absolute cell count per ml blood (bottom, n = 5). (D) Confocal micrographs of Myh9 +/− and Myh9 −/− BMDMs, CCL2-stimulated and ICAM-bound, followed by HuR immunostaining for translocation analysis. Bar, 10 µm. (E) Quantification of percentage of cells in D with HuR nuclear-to-cytosolic translocation (**, P < 0.002; n = 3). (F) VEGF-A mRNA decay assays, using the indicated BMDMs, adhered to immobilized ICAM-1 or Fcγ fragment control in the presence or absence of CCL2 for 30 min, after which transcription was arrested with 10 mg/ml actinomycin (time 0), and RNA collected at the noted time points for VEGF-A mRNA quantification by RT-qPCR (**, P = 0.0001; n = 5). (G and H) Laser Doppler scan images (G) of flow in the ischemic (I) and contralateral control (C) hind limb of Myh9 +/− and Myh9 −/− mice at the indicated time points before and after femoral artery ligation with quantitative blood flow analysis (H; **, P < 0.0006; n = 7). (I) Real-time PCR quantification of VEGF-A mRNA in thigh (adductor) muscle tissue obtained 3 d after femoral artery ligation (**, P = 0.003; n = 4). Animal data are from 3 independent experiments, each with 2–3 mice per treatment group. Remaining data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Article Snippet: Anti-Rac2 mouse monoclonal antibody was purchased from Proteintech.

Techniques: Translocation Assay, Western Blot, Isolation, Cell Counting, Immunostaining, Control, Quantitative RT-PCR, Ligation, Real-time Polymerase Chain Reaction