Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 3. Caveolin-1 interacts with ICAM-1 primarily through filamin A. HeLa cells treated with siRNA for filamin A, filamin B, caveolin-1 (Cav1), or control (ctrl) were used in a pull-down (PD) assay using the biotinylated pep- tide encoding the intracellular domain of ICAM-1. Cell lysates and isolated proteins were analyzed by Western blot for caveolin-1, filamin A, filamin B, and tubulin (protein loading control).

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Control, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 6. Filamin mediates lateral membrane mobility of ICAM-1. FRAP analysis in combination with confocal imaging was used to study membrane dynamics of GFP-CAAX as a control (A) and ICAM-1-GFP (B) in HeLa cells trans- fected with control (gray line) or filamin B (dark line) siRNA. Western blot in A shows expression analysis of filamin B. Tubulin is included as a loading con- trol. Dashed lines indicate pre-bleaching intensity set at 100%. A, the fluores- cence recovery of GFP-CAAX was not affected by reduced expression of fil- amin B. Graph is representative of four independent experiments. B, images show ICAM-GFP distribution in control siRNA or filamin B siRNA-treated cells. Bar, 20 m. In control siRNA-treated cells, ICAM-1-GFP recovered to 70% within 2.5 min (gray lines), whereas filamin B-deficient cells showed recovery to maximal 40% (dark lines). The graph is representative of four independent experiments. C, mobile fractions of both experiments are calculated and show that filamin B siRNA significantly reduces the mobile fraction of ICAM- 1-GFP but not GFP-CAAX. Data are mean S.E. of four independent experi- ments;*,p0.01.D,measurementoftheslope(K)oftherecoverydepictedin A and B shows that reduction of filamin B did not affect the speed of motility ofICAM-1-GFPorofGFP-CAAXintheplasmamembrane.DataaremeanS.E. of four independent experiments.

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Membrane, Imaging, Control, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 7. Filamin B mediates recruitment of ICAM-1. A, ICAM-1 antibody-coated beads were incubated with ICAM-1-GFP expressing HeLa cells and the recruitment of ICAM-1-GFP to the beads was recorded for 20 min using time lapse confocal imaging. Time in minutes is shown above the images. Cells were treated with control siRNA or filamin B siRNA, as indicated on the left. Still images in A show ICAM-1-GFP recruitment around ICAM-1 antibody-coated beads in siRNA control cells after 4 to 8 min (open arrowheads in upper panels), whereas ICAM-1-GFP recruitment occurs not before 20 min in cells with reduced filamin B expression (lower images). Bar, 10 m. Lower panels show line-scan of the fluorescent intensity of ICAM-1-GFP surrounding the bead, indicated by the white dashed bar. B, quantification of the number of cup structures, formed around adherent beads on cells treated or not (ctrl) with siRNA to filamin B. *, p 0.001.

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Incubation, Expressing, Imaging, Control

Journal: Journal of Biological Chemistry

Article Title: Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

doi: 10.1074/jbc.m804888200

Figure Lengend Snippet: FIGURE 8. Filamin B regulates ICAM-1-mediated adhesion and transmi- gration under static and flow conditions. A, reduced filamin B levels reduced the adhesion of ICAM-1 antibody-coated beads to ICAM-1-GFP expressing HeLa cells. Beads were counted per field of view, 20 fields per experiment were counted and three independent experiments have been carried out. HeLa cells were either not treated, or transfected with ICAM-1- GFP and treated with control siRNA (ctrl) or filamin B siRNA as indicated. Data are mean S.E. of a representative experiment performed in quadruple. *, p 0.05. B, differentiated HL60 cells were allowed to migrate across a HeLa ICAM-1-GFP monolayer in a Transwell system to 50 ng/ml SDF-1. Percentage of migration was determined as described under “Experimental Procedures.” HeLa cells were either not treated or transfected with ICAM-1-GFP and treated with control siRNA (ctrl) or filamin B siRNA as indicated. Data are mean S.E. of a representative experiment performed in quadruple. *, p 0.05. C, differentiated HL60 cells were perfused over monolayers of TNF-- treated primary human endothelial cells. Migration was visualized by adhe- sive cells crossing the monolayer thereby changing from a bright to a dim appearance (arrowheads). D, differentiated HL60 cells were flown over mono- layers of TNF--treated primary human endothelial cells. Left panel shows quantification of leukocyte migration across endothelial monolayers trans- fected with filamin B siRNA (si-filamin B), compared with migration across siRNA control treated endothelial cells (ctrl). Right panel shows quantification of adhesion of HL60 cells to endothelial cells treated with siRNA to filamin B. Data represent averages of duplicates from one of two independent experiments.

Article Snippet: Transmigration under Flow—Primary HUVECs were cultured to confluence in EGM2 (Cambrex) on fibronectin-coated glass coverslips transfected with either control or filamin B siRNA according to manufacturer’s protocol using siRNA transfection reagent (Santa Cruz, Santa Cruz, CA).

Techniques: Expressing, Transfection, Control, Migration

Journal: The American Journal of Pathology

Article Title: Expression and Potential Role of Fas-Associated Phosphatase-1 in Ovarian Cancer

doi: 10.1016/s0002-9440(10)64084-9

Figure Lengend Snippet: Figure 2. Comparison of FAP-1 protein levels in ovarian cancer cell lines. Lysates from cell lines (100 g total protein) were subjected to SDS-PAGE/ immunoblot assay using rabbit polyclonal no. 1730. Jurkat and HEK 293 cells served as negative and positive controls, respectively. A nonspecific band is also observed (asterisk), serving as a loading control. Densitometrical anal- ysis was performed using a Multi Image light cabinet and the ChemiImager software version 4000 yielding values based on integrated density (#). Data were arbitrarily normalized relative to FAP-1 levels of HEK 293 cells that were included on all blots as an internal control.

Article Snippet: To quantify immunoblotting results, films were analyzed densitometrically using a Multi Image Light Cabinet and the ChemiImager software version 4000 (Alpha Innotech Corporation, San Leandro, CA).

Techniques: Comparison, SDS Page, Western Blot, Control, Software