1. ICAM-1 forms distinct complexes with filamin B, α-actinin-4 and cortactin.
ICAM-1 forms distinct complexes with filamin B, α-actinin-4 and cortactin. (A) Western blots show that depletion (by siRNA, si-) of filamin B, cortactin or α-actinin-4 in TNFα-treated HUVEC did not affect binding of the other adaptors to ICAM-1 (total cell lysate in supplementary material Fig. S1A). Bar graphs show quantification of the interactions (n = 3). (B) Sequence alignment of the intracellular ICAM-1 domain from human, mouse and rat (left panel) and the structure model of the human intracellular ICAM-1 domain (right panel). Mutated residues Lys519 and Lys524 are indicated by arrows and in stick representation. TM, transmembrane. (C) Full-length human wild-type (WT) ICAM-1 and indicated mutants were expressed in ICAM-1-deficient HeLa cells, prior to analysis of binding of endogenous filamin B, cortactin and α-actinin-4 to clustered ICAM-1. Western blots show expression in the total cell lysate (TCL, left panel) and binding to ICAM-1 [pull-out, right panel; quantification, bar graph (n = 3)]. (D,E) Neutrophils were added to TNFα-treated HUVECs transfected with ICAM-1-K519A-K524A. Cells were fixed after 25 min and stained for HA-tagged ICAM-1-K519A-K524A and F-actin (phalloidin). Neutrophils migrating across HUVECs expressing the ICAM-1 K519A-K524A mutant (green) show (D) less polarization, as deduced from the F-actin distribution (images represent one out of two experiments) and (E) less spreading (as calculated in Fig. 1B; n = 2, 10 neutrophils per group). Scale bar: 10 µm. Data are mean±s.e.m. *P<0.05; **P<0.01, ***P<0.001 (Student's t-test).
Antje Schaefer et al. J Cell Sci 2014;127:
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