FLNa regulates FA disassembly. (A) BT-20 cells expressing empty retroviral particles (PSR) or FLNa-specific shRNA (PSR-FLNa) were seeded on fibronectin- or collagen-coated dishes and grown at 70% confluence. Total protein extracts were probed using antibodies against the indicated proteins. Bar graph shows quantification of ratio of phosphorylated/total proteins (mean ± SD) from four independent experiments (*, P < 0.05). (B) Serum-starved control (PSR) and FLNa-silenced (PSR-FLNa) BT-20 cells were incubated with 10 µM nocodazole (NZ) for 4 h. Control untreated cells and treated cells at the indicated times after nocodazole washout were fixed and immunoassayed with anti–paxillin-pY118 or anti-FLNa. Three independent experiments were performed. Bar, 20 µm. (C) Quantification of FA areas using Volocity software. Data are expressed as the area of FAs per DAPI-stained nucleus and were obtained from at least 20 cells per condition. Quantifications show mean ± SD from three independent experiments as in B (*, P < 0.01). (D) Representative Western blots of FAK and paxillin during FA disassembly. BT-20 cell lysates were collected at the indicated times as in B and immunoblotted with anti–FAK-pY397 or anti–paxillin-pY118. Bar graphs represent the densitometry result as percentage of density of PSR control at 0 min (which is 100%) from four independent Western blots. MM, molecular mass.