Transfection and siRNA knockdown. Gene silencing was performed using ON-TARGET plus SMARTpool selleck chemical Nilotinib HuR siRNA, which contains a mixture of four siRNAs that target HuR (30 nM) purchased from Dharmacon, as we have described (5). EC transfection was performed using the Human EC Nucleofector Kit (Amaxa) following the manufacturer’s instructions. Transfection efficiency was 70�C90% as assayed by dual transfection of a GFP reporter plasmid (14). Lysates were immunoblotted for HuR 72 h posttransfection, and RNA was extracted 72 h posttransfection. HuR cDNA was purchased in an expression vector (Origene Technologies) and was overexpressed by transfection of 2 ��g/106 ECs using the Nucleofector kit as described for siRNA. Monocyte adhesion assay. Adhesion was assayed as described (16).

Briefly, hCaECs were cultured on glass coverslips at a density of 6 �� 105 cells/chamber. Confluent ECs were treated with IL-19 (100 ng/ml) for 16 h, then in the presence or absence of TNF-�� (10 ng/ml) for an additional 6 or 24 h, followed by extensive washing with PBS. THP-1 human monocytes were purchased from the American Type Culture Collection (catalog no. TIB-202) and cultured according to vendors instructions. THP-1 monocytes (5 �� 105 cells/well) were labeled with 2��,7��-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM, 10 ��M, Sigma) and incubated with hCaECs for 30 min at 37��C. Unbound THP-1 cells were removed by gently washing them twice with PBS, and adherent cells were fixed with 4% formalin, photographed by an inverted fluorescent microscope (Eclipse TS-100, Nikon), and counted per high-power field (HPF).

For some experiments, anti-IL-20R�� (catalog no. MAB11762, R&D) or negative control antibody was added to ECs 2 h before addition of IL-19 to neutralize the IL-19 receptor. Results are expressed as percentages of the controls and represent means �� SE from triplicate experiments. Intravital microscopy, animal care. Leukocyte rolling and adhesion were assayed in mesenteric postcapillary venules by intravital microscopy as described previously (27). Mice (20 g body wt) were then injected intraperitoneally with 10.0 ng/g IL-19 followed 16 h later by intraperitoneal injection with 20.0 ng/g body wt TNF-��. Rolling and adhesion were quantitated 4 h following TNF-�� injection. Wild-type C57BL/6 mice were injected with 120 mg/kg pentobarbital sodium by intraperitoneal injection. Depth of anesthesia was monitored by toe-pinch and blood pressure. Three to four straight, unbranched segments of postcapillary venules with lengths of >100 ��m and diameters Brefeldin_A between 25 and 40 ��m were studied in each mouse using an Eclipse FN1 Microscope (Nikon), and the images were recorded and analyzed on A WIN XP Imaging Workstation.