Fig. 4. Focal adhesion and actin organization is disturbed in IKAP-depleted MEFs. (A) IKAP-depleted (m2) or control (scr) MEFs were stained for paxillin and vinculin at 1.5 hours after plating and for actin at 24 hours. Human IKAP or FD-IKAP was introduced to some of the murine IKAP-depleted cells as indicated. (B) Quantification of the number of cells with organized paxillin staining (means ± s.d.; n=3; ^**P<0.01). (C) Quantification of the percentage of cells with organized actin cytoskeleton (means ± s.d.; n=3; ^**P<0.01). (D) Western blot analysis of protein levels in MEFs transduced with designated IKBKAP shRNA constructs (left panel) or HeLa cells transiently transfected with siRNA oligonucleotides (right panel) for 48 hours. (E) The soluble (S; G-actin) and insoluble (I; F-actin) actin content of HeLa cells transiently expressing H1 IKBKAP siRNA or scr control oligonucleotide was assessed by western blotting. Triton X-100 soluble and insoluble proteins were extracted as described in the Materials and Methods, and lysates were analyzed for IKAP and β-actin. The quantification was made by ImageGauge software. The quantification is representative of two independent experiments. (F) Paxillin and beclin 1 protein levels in western blot analysis of FD-patient fibroblasts and fibroblasts from a healthy control individual. Scale bars: 20 µm.