Actin-binding defective mutation in filamin A impairs integrin outside-in signaling. (A) Analysis of αIIbβ3 outside-in signaling in HEL cells transfected with EGFP vector (V), EGFP-filamin (WT), and EGFP-filamin F99A/L104E (FA/LE) and simulated with 0.5 mM Mn2+ in the presence of 25 μg/mL fibrinogen in suspension. At indicated time points, HEL cells were lysed and analyzed using western blotting, with antibodies recognizing phosphorylated Src Tyr416, phosphorylated FAK Tyr397, Src, FAK, and GFP. Band intensity of phosphorylated Src (B) and phosphorylated FAK (C) was quantified using imageJ. Data are averages ± SEM for 2 independent experiments. ∗P < .05. (D) Regulation of cell migration by filamin. Representative images of HEK293T cells transfected with pmCherry, pmCherry-WT-Filamin, pmCherry-F99A/L104E-Filamin respectively at time 0 and 24 hours after a scratch was introduced in the monolayer with a 20 μl pipette tip. (E) The average area of the open wound of the indicated conditions at 24 hours after a scratch was introduced. The area was shown as the percentage of wound area at time 0. Data are averages ± SEM for 6 independent experiments. ∗∗∗P < .001. FAK, focal adhesion kinase; HEL, human erythroleukemia cell; Src, proto-oncogene tyrosine kinase.
