Cleavage of FLNa regulates FA turnover and cell invasion. (A) BT-20 siFLNa cells were transfected with construct either expressing GFP-tagged FLNa WT (GFP–FLNa-WT) or GFP-tagged FLNa mutant resistant to calpain cleavage (GFP–FLNa-Δ) and assayed for nocodazole (NZ)-based FA disassembly. Cells were untreated (control) or incubated with 10 µM nocodazole for 4 h. Cell lysates were collected at the indicated times and immunoblotted with anti–FAK-pY397 or anti–paxillin-pY118. (B) BT-20 siFLNa cells transfected with either GFP–FLNa-WT or GFP–FLNa-Δ were cultured on 10-cm dishes and stimulated or not with degraded type I collagen (De.Collagen) for the indicated times. Where indicated, cells were treated with 50 µM of calpain inhibitor ALLN (22 h). Lysates were prepared at the indicated time points and analyzed by Western blotting using the corresponding antibodies. Control indicates unstimulated. NT, N terminus. (C) Control BT-20 cells, BT-20 siFLNa cells, and BT-20 siFLNa cells expressing GFP–FLNa-WT or GFP–FLNa-Δ were placed in the top layer of Boyden migration chamber, and after 48 h, cells migrated through the chamber to the bottom membrane were measured. The graph represents the mean ± SD from four independent experiments (*, P < 0.005). (D) FLNa-silenced BT-20 cells were transfected with construct either expressing EGFP-tagged FLNa 14–16 repeats (EGFP-FLNa (14–16)) or expressing EGFP alone, stimulated or not stimulated with degraded type I collagen. Cell lysates were immunoblotted with the indicated antibodies. Bar graph shows quantification of cleavage of FAK (mean ± SD) from three independent experiments. MM, molecular mass.