Fig. 4.

Annexin A2 and protein S100-A10 regulation of HMW-HA-induced filamin A/B recruitment to CEM and human EC barrier enhancement. A: immunoblot analysis of HMW-HA-treated (100 nM, 5 min) or untreated human EC lysates from scramble siRNA, annexin A2 siRNA, protein S100-A10 siRNA, or annexin A2 + protein S100-A10 siRNA transfection using anti-filamin A (a), anti-filamin B (b), or anti-caveolin-1 (c) antibody. Experiments were performed in triplicate, each with similar results, and representative data are shown. Silencing annexin A2 and protein S100-A10 blocks HMW-HA-induced filamin A and filamin B recruitment to CEM. B: EC were treated with no siRNA (control), scramble siRNA, filamin A siRNA, or filamin B siRNA for 48 h. EC lysates were obtained and run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-filamin A (a), anti-filamin B (b), or anti-actin (c) antibody. Experiments were performed in triplicate, with highly reproducible findings, and representative data are shown. C: percent inhibition of maximal HMW-HA-induced TER response. EC were plated on gold microelectrodes and treated with scramble siRNA (control), annexin A11 siRNA, filamin A siRNA, filamin B siRNA, or filamin A + filamin B siRNA for 48 h. After EC were serum-starved for 1 h, 100 nM HMW-HA was added. Values represent pooled TER data ± SE at 30 min after addition of agonist from 3 independent experiments.