Fig. 3

Expression of LD2-LD3-LD4 blocks kinase-dependent functions of FAK, downstream of integrin activation a) Representative Western Blots and quantification from control cells and cells expressing GFP LD2-LD3-LD4, indicating the phosphorylation status of FAK Tyr 397 and Tyr 576 and Paxillin Tyr 31. Quantification of the ratio of phosphorylated FAK over total FAK shows reduction of the phosphorylation at both Tyr 576 (0.13 ± 0.024 in control, 0.074 ± 0.005 in GFP LD2-LD3-LD4 expressing samples) and Tyr 397 (0.22 ± 0.0073 in control, 0.13 ± 0.015 in GFP LD2-LD3-LD4 expressing samples). Paxillin Tyr 31 phosphorylation is also reduced, as indicated by the ratio of phosphorylated over total paxillin (0.24 ± 0.08 in control, 0.11 ± 0.04 in GFP LD2-LD3-LD4 expressing samples). b) Confocal images of PFA-fixed HeLa cells transfected with GFP LD2-LD3-LD4 and immunostained for Paxillin and phosphorylated Paxillin (pY31). Phosphorylated Paxillin signal in control and GFP LD2-LD3-LD4 expressing cells (marked with asterisk) is presented in the middle panel as an intensity color-coded image. Quantification of the % change in the mean intensity of phosphorylated (pPaxillin) to total Paxillin reveals significant reduction of Paxillin phosphorylation at FAs in cells expressing GFP LD2-LD3-LD4 (27.93 ± 0.83, n = 331 FAs from 24 cells) compared to control cells (100 ± 2.92, n = 371 FAs from 25 cells). c) Confocal images of PFA-fixed HeLa cells transfected with GFP LD2-LD3-LD4, immunostained against phosphorylated tyrosine (pY20) and Vinculin. Intensity of tyrosine phosphorylation in control and GFP LD2-LD3-LD4 expressing cells (marked with asterisk) is presented in the middle panel in a color-coded image. Quantification of the % change in the mean pY20/Vinculinintensity reveals a 3.4-fold decrease in total phosphorylation at FAs in cells expressing GFP LD2-LD3-LD4 (29.29 ± 2.12, n = 414 FAs from 30 cells) compared to control cells (100 ± 4.44, n = 435 FAs from 30 cells). d) Confocal images of PFA-fixed cells transfected with GFP LD2-LD3-LD4 and immunostained against active β1 Integrin and Vinculin. Active β1 Integrin signal in control and GFP LD2-LD3-LD4 expressing cells (marked with asterisk) is presented in the middle panel as an intensity color-coded image. Quantification of the % change in the mean active β1 Integrin/Vinculin intensity shows that expression of GFP LD2-LD3-LD4 does not affect integrin activation at FAs (100 ± 11.18, n = 346 FAs from 30 control, compared to 97.10 ± 3.98, n = 457 FAs from 30 expressing cells). Scale bars: 10 μm. The error bars represent standard error of the mean (S.E.M). ***; p < 0.001