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. 2013 Mar 26;8(3):e60528. doi: 10.1371/journal.pone.0060528

Figure 4. Dispensable role of I-BAR domain for podosome formation.

Figure 4

(A) Restoration of podosome formation by the expression of the wild-type (WT) or BPM mutant of IRSp53. NIH-Src cells were transfected sequentially with IRSp53 siRNA and the expression vectors, and were stained with rhodamine-phalloidin (red) to visualize actin filaments and with anti-IRSp53 (blue). Scale bar, 20 µm. (B) Percentage of cells with podosomes in (A). Data are means±SD from three independent experiments. More than 100 cells were counted in each determination. *P<0.05 (Student's t test). (C) NIH-Src cells were transfected simultaneously with the FLAG-tagged constitutively active form of Rac (Rac1 G12V) and the GFP-tagged I-BAR domain of IRSp53. The R11E/Q23E mutant of I-BAR, which is defective in Rac binding, was used as a negative control for Rac binding [16]. Anti-FLAG immunoprecipitates were then subjected to immunoblot analyses with the indicated antibodies.