Human ROBO4 Antibody

Detection of ROBO4 in HUVEC cells by Flow Cytometry

HUVEC cells were stained with Goat Anti-Human ROBO4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2366, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram) followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108). View our protocol for Staining Membrane-associated Proteins.

Detection of ROBO4 by Western Blot

ROBO4 suppresses PTGS2 expression by inhibiting RAC1-JNK signaling. A Effect of ROBO4 on active RAC1 in ECs. Cell extracts were prepared from HUVECs treated with siRNA and TNF and used for measurements of active RAC1 using a G-LISA kit (n = 7). b, c Regulation of JNK phosphorylation by ROBO4 and RAC1. HUVECs were transfected with siRNA and treated with TNF in the presence or absence of pretreatment with NSC23766 (100 μM). Expression levels of p-JNK, JNK, GAPDH, and ROBO4 in the cells were analyzed by western blotting. Relative p-JNK levels were quantified using ImageJ software and calculated by normalizing with JNK levels (n = 5 (b); n = 4 (c)). d, e Contribution of RAC1 and JNK to ROBO4 knockdown-induced PTGS2 upregulation. HUVECs were transfected with siRNA, pretreated with NSC23766 (100 μΜ) or SP600125 (10 μM), and treated with TNF. Expression of PTGS2 and GAPDH were measured by qRT-PCR (n = 5). Data are expressed as the mean ± standard error of the mean (a–e). *P < 0.05, **P < 0.01, and ***P < 0.001, P values were calculated with a two-way analysis of variance followed by Bonferroni’s test between control and ROBO4 siRNA groups at each time point (a) or Tukey’s test (b–d). Non-specified P values in the graph are not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38762541), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ROBO4 by Western Blot

ROBO4 suppresses PTGS2 expression by inhibiting AP-1 and catenin beta-1.a, b Effect of ROBO4 on TNF-induced PTGS2 expression in ECs. HUVECs were treated with siRNA and TNF, and expression of PTGS2, Robo4, and GAPDH mRNA and proteins were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (a) and immunoblotting (b), respectively. PTGS2 and Robo4 levels were normalized to GAPDH levels (n = 4). c, dPTGS2 expression in HUVECs treated with siRNA, TNF, and AP-1 inhibitor (SR 11302) (c) or CTNNB1 siRNA (d) (n = 9 or 7, respectively). PTGS2 and GAPDH mRNA levels were measured by qRT-PCR. e Effect of ROBO4 on subcellular localization of catenin beta-1. Cytoplasmic and nuclear protein extracts were prepared from HUVECs treated with siRNA and TNF, and analyzed for catenin beta-1, GAPDH, and Lamin B1 expression by western blotting. Expression levels of catenin beta-1, GAPDH and Lamin B1 were quantified using the ImageJ software (n = 4). Relative nuclear catenin beta-1 levels were calculated by normalizing to Lamin B1 levels. Data are expressed as the mean ± standard error of the mean (a–e). *P < 0.05, **P < 0.01, and ***P < 0.001, P values were calculated with two-way analysis of variance followed by Tukey’s test (a–d) or Welch’s t-test (e). Non-specified P values in the graph are not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38762541), licensed under a CC-BY license. Not internally tested by R&D Systems.