Search for author "Zhaohua Cai"

Figure 3. Vascular ECs express the FKN receptor CX3CR1. a, The cellular lysates from cultured HCAECs and HUVECs and fresh human leukocytes were used for Western blots, using anti-human FKN and CX3CR1 antibodies. Both types of ECs (left 2 lanes) express both FKN and CX3CR1. Macrophages and neutrophils express CX3CR1 but not FKN itself. CX3CR1 in ECs has two 50-kDa bands as well as a 40-kDa band. Macrophages (MΦ) (fourth lane) express only a 40-kDa CX3CR1 protein, and neutrophils (PMNs) (right lane) also express it but in lesser amounts. b, RT-PCR assay for CX3CR1 mRNA. A pair of primers from the V28 cDNA sequence–spanning exons 2 and 4 was used to amplify reverse transcript products from total RNA from ECs and leukocytes. The RT-PCR products were run on 5% polyacrylamide gel and stained with ethidium bromide. ECs and leukocytes both express CX3CR1 mRNA. However, PMNs express less than ECs and MΦ. c, Confocal microscopic images of an HUVEC. Cultured HUVECs were fixed with methanol, blocked with goat serum, and incubated with anti-human CX3CR1 antibody. After washing, cells were incubated with secondary antibody labeled with green fluorescence. Top, Confocal scan sections of a single HUVEC. Bottom, Image of a single-scan section (2.24 μm) through the cell. The green staining, representing CX3CR1, is located primarily on the cell membrane and partially extends to the cytoplasm. The nucleus is stained blue with DAPI (2′,6′-diamidino-2-phenylindole).Show Less

Figure 5. FKN increases adhesion of PMNs to HUVECs, and the increased adhesion is blocked by anti–ICAM-1 antibody. a, Fluorescein isothiocyanate (FITC) and myeloperoxidase (MPO) activities, representing adherent PMNs, increased significantly in control cells during s-FKN simulation (**P<0.001) but not in ECs with CX3CR1 knockdown. b, ICAM-1 antibody blocked PMN adhesion to HUVECs during s-FKN stimulation, indicating that the increase in PMN adhesion induced by s-FKN is ICAM-1 dependent.Show Less

Figure 7. a, Immunoprecipitation assay for Stat5 isoforms. HUVECs were exposed to 10 nmol/L s-FKN for 10 minutes, and the cells were lysed in radioimmunoprecipitation assay buffer.17 Anti-Stat5α and -Stat5β antibodies were then added, and the tubes were rotated overnight at 4°C. The complexes were pulled down with protein A agarose (Invitrogen) and run on NuPAGE gel for immunoblotting using anti–phosphorylated tyrosine Stat5 and anti-Stat5 antibodies. IP indicates immunoprecipitation. The Stat5 that became phosphorylated was the α isoform. b, Electrophoretic mobility-shift assay and competition assays show that Stat5α binds to the GAS element in the ICAM-1 promoter. ECs were exposed to 10 nmol/L s-FKN or control (C) medium for 10 minutes, and the cells were harvested to isolate nuclear protein (NE). For competition assays, unlabeled GAS, anti-Stat5 antibody, or anti-Stat5α or -β antibodies were used.Show Less