Fig. 8

LD2-LD4 expression interferes with the interaction between FAK and paxillin. Representative Western Blot from inducible GFP LD2-LD4 stable Hela cells indicating the expression levels of GFP LD2-LD4 compared to endogenous paxillin, upon induction with 10 μg/ml doxycycline for 24 h. b) Western blot showing immunoprecipitated GFP, from Hela cells stably expressing GFP or GFP LD2-LD4 (from inducible GFP LD2-LD4 Hela cells, induced or not with 10 μg/ml doxycycline for 24 h), blotted for GFP and FAK. FAK co-precipitation is observed in cells inducibly expressing GFP LD2-LD4. c) Confocal images of PFA-fixed inducible GFP LD2-LD4 Hela cells, immunostained for FAK and Vinculin. Expression of GFP LD2-LD4 upon treatment with 10 μg/ml doxycycline for 24 h (bottom panel) leads to decrease in the intensity of FAK at FAs, unlike Vinculin. d) Quantification of the % change in the mean FAK/Vinculin intensity at FAs reveals ~ 67% reduction in the FA-localized FAK in cells inducibly expressing GFP LD2-LD4 (32.28 ± 1.664 n = 197 FAs from 27 cells) compared to control non-induced cells (100 ± 4.01 n = 211 FAs from 28 cells). e) Representative western blots from isolated FAs of inducible GFP LD2-LD4 Hela cells, non-induced (left lane) or induced with 10 μg/ml doxycycline for 24 h, blotted for FAK, Paxillin and Vinculin. Quantification of FAK/Actin (0.7252 ± 0.2068 in non-induced, 0.3583 ± 0.1684 in induced cells), Paxillin/Actin (3.075 ± 0.7076 in non-induced, 5.484 ± 2.322 in induced) and Vinculin/Actin (0.2735 ± 0.1407 in non-induced, 0.3610 ± 0.1156 in induced) ratios, show reduction of FAK at the FAs of induced cells, unlike Paxillin and Vinculin which display increased FA-localization, verifying the results of IF experiments. This is further confirmed by quantification of the normalized FAK/Paxillin ratio in induced (0.05682 ± 0.02397) and non-induced (0.2320 ± 0.04788) cells, indicating 75% reduction in FA-localized FAK in cells expressing GFP LD2-LD4. f) Western blot of immunoprecipitated FAK from induced (24 h treatment with 10 μg/ml doxycycline) and non-induced GFP LD2-LD4 Hela cells, blotted for FAK and paxillin. The amount of co-precipitated paxillin is reduced in induced cells expressing GFP LD2-LD4. Non-induced GFP LD2-LD4 cells incubated with no antibody were used as negative control. Scale bars 10 μm. The error bars represent standard error of the mean (S.E.M). *; p < 0.0.05