Fig. 7

The LD2 and LD4 motifs of paxillin, are sufficient for displacement of FAK from FAs a) Schematic representation of LD2-LD3-LD4 ΔLR, composed of amino acids 139–279 of Paxillin fused to GFP; and LD2-LD4, composed of LD2 and LD4 motifs of paxillin, amino acids 139–162 and 261–279 respectively, joined together by a 30 amino acid-long flexible linker and fused to GFP. b) Representative Western Blot showing expression of stable proteins, encoding GFP fused LD2-LD3-LD4 ΔLR and LD2-LD4, in HeLa cells (expected molecular weight ~ 44 kDa and ~ 34 kDa respectively). c) Widefield images of HeLa cells, control or transiently transfected with GFP-fused LD2-LD3-LD4, LD2-LD3-LD4 ΔLR or LD2-LD4 and immunostained for FAK and Vinculin. Expressing cells are marked with an asterisk. Control cells display strong localization of FAK at FAs unlike cells expressing GFP-fused LD2-LD3-LD4, LD2-LD3-LD4 ΔLR or LD2-LD4. d) Quantification of the % change in the mean FAK/Vinculin intensity at FAs in control cells (100 ± 4.56, n = 335 FAs from control cells) and cells expressing GFP LD2-LD3-LD4 (26.33 ± 1.46, n = 163 FAs from 16 low-expressing and 21.2 ± 2.73, n = 147 FAs from 15 high-expressing cells), GFP LD2-LD3-LD4 ΔLR (33.04 ± 2, n = 150 FAs from 16 low-expressing and 24.32 ± 1.84, n = 133 FAs from 14 high-expressing cells) or GFP LD2-LD4 (30 ± 1.3, n = 140 FAs from 15 low-expressing and 21.22 ± 1.22, n = 225 FAs from 17 high-expressing cells). Both GFP LD2-LD3-LD4 ΔLR and GFP LD2-LD4 displace FAK from FAs in a dose dependent manner and as efficiently as GFP LD2-LD3-LD4 does. e) Quantification of the % change in the mean FAK/Vinculin intensity at FAs in control cells (100 ± 6.5, n = 119 FAs from 15 cells) and cells expressing GFP LD2-LD3-LD4 (36.65 ± 3.4, n = 140 FAs from 14 cells), or GFP LD2-LD4 with either a 30 amino acid linker (38.3 ± 2.35, n = 100 FAs from 14 cells), 25 amino acid linker (58.47 ± 2.44, n = 117 FAs from 15 cells) or a 15 amino acid linker (68.69 ± 3.31, n = 110 FAs from 15 cells) It is evident that the 30 amino acid long linker displays equivalent efficiency to displace FAK from FAs to LD2-LD3-LD4. Scale bars 10 μm. The error bars represent standard error of the mean (S.E.M). ***; p < 0.0001