Fig 2: Western immunoblotting of insoluble (A) and TBS-soluble (B) Filamin A (FLNA) migrating at approximately 250 kDa. Actin (42 kDa) is shown in soluble fractions. Subjects are identified by group. ADD, AD dementia.

Fig 3: The expression of Filamin A and the activation of Rac 1 and FAK (Tyr 397) are important for androgen induced LNCaP migration. In (A–E), siRNA Alexa Fluor 488, control siRNA (Ctrl siRNA) or siRNA FlnA (FlnA siRNA) or siRNA AR (AR siRNA) were used as in “Methods” section. (A) After transfection, cellular lysates were obtained and FlnA (left) and AR (right) expression were analyzed by WB using the appropriate antibodies. Transfected and quiescent cells, unstimulated or stimulated as indicated, were used for migration (B–D) or invasiveness assays (C–E) for seven h or 24 h, respectively. Migrating (B–D) or invading (C–E) cells were scored as in “Methods” section and data expressed as fold increase. Means and SEM are shown. In (F), proteins lysates were used for Rac pull down assay using a commercially available kit, as described under “Methods”. The WB with anti-Rac antibody revealed the total amount of Rac expressed in the corresponding lysates (upper panel) and the eluted Rac (Rac-GTP; lower panel). In (G), lysate proteins were analyzed for Fak activation (P-Fak Y397), using the anti-P-Tyr397Fak antibody. The filter was stripped and re-probed using anti-Fak antibody (upper section). The WB for tubulin expression in lysate proteins was finally done, as loading control. In (A–E) results are representative of 3 different experiments and when indicated n represents the number of experiments. In (B–E), in ctrl siRNA transfected cells, R1881 increases significantly the cell migration (B,D) or BrdU incorporation (C,E).

Fig 4: F-actin density reduction by an FLNa deficiency at the NK IS. (A) NKL cells nucleofected with control or FLNa-specific siRNAs were incubated with Cell tracker orange CMTMR-loaded 721.221 cells (red) for 15 min, fixed, permeabilized, and stained for F-actin (green). F-actin accumulation at the IS between NKL and 721.221 cells was analyzed by confocal imaging. The image is representative of 12 conjugates analyzed. (B) F-actin accumulation at the NK IS was estimated using a reconstruction of serial confocal images acquired via the z-axis and a single compressed image of the NKL and 721.221 cells conjugate shown in (A). A summary graph is presented. The horizontal line represents the mean, and the vertical bar indicates the SD; ***P < 0.001. Scale bar, 5 µm. Results are representative of three independent experiments.

Fig 5: The effects of PN/integrin ß1 on association of Pak1 with FLNA, a substrate for Pak1 kinase activity in VIC lysates. (A,B) VICs were transfected with lentivirus encoding Myc-FLNA for 48 h and then serum starved for 24 h. They were then either untreated or treated with 1 µg/ml of PN for 1 h. (A) Cell lysates were immunoprecipitated without (lane 1, control IgG) or with an antibody against Myc (for FLNA) and immunoblotted with antibodies against Pak1, Myc, or FLNA. (B) VICs were pretreated with cytoskeleton disrupter 2 µM cytochalasin D (CytoD) for 30 min and then treated with 1 µg/ml PN for 1 h (lane 4) and compared with VICs treated with PN alone (lane 3). Cell lysates were immunoprecipitated with an antibody against Myc (for FLNA) and immunoblotted with antibodies against Pak1, Myc, or for FLNA in WCL. Lane 1 is for control IgG. (C) Upper Panel: VICs were cotransfected with the FLNA S2152A mutant, or DN Pak1 containing Pak1 autoinhibitory domain (residues 83–149), or DN Cdc42, or 5 µg/ml of a5 and ß1 integrin blocking antibodies for 2 h. After 48 h, the cells were serum starved, or treated with or without 1 µg/ml of PN as in the experiment in Figure 2G. Pak1 was immunoprecipitated, and a nonradioactive kinase traditional in-gel phosphorylation assay was done using GST fusion proteins of FLNA as substrate. Isotype control IgG was used as control. For details, see the “Materials and Methods” section. Lower Panel: Bars represent densitometric ratios of active phospho pFLNA (S2152)/total FLNA in the lysates. (D) In vitro Pak1 kinase assays from the experiment in panel (C) were further confirmed by assaying using the nonradioactive assay kit (ab138879, Abcam), which is based on the monitoring of ADP formation (see “Materials and Methods” section for details). Data are presented as fold increase in Pak1 kinase activity with respect to control. The data in the experiments in panel (A–D) are representative of =3 independent experiments. The values in experiments (A–B) were determined by densitometry. The values derived from the control cells were designated as 1.0. Error bars are reported as means ± SD of =3 separate experiments.