Fig. 3. Annexin A2 depletion inhibits insulin-induced actin rearrangements. (A) Downregulation of annexin A2 does not affect the general tyrosine phosphorylation in BHK-IR cells. Cells were nontransfected, transfected with control (mock) or annexin A2-specific siRNAs, serum-starved and insulin-stimulated for 30 minutes as indicated. Total cellular lysates were then immunoblotted for phosphotyrosine (-pY). The blot was stripped and reprobed for the IRβ-subunit (-IRβ) and annexin A2 (-Anxa2). (B) Effect of RNAi-mediated downregulation of annexin A2 on insulin-induced actin rearrangements. BHK-IR cells, grown on coverslips, were mock transfected or transfected with annexin A2 siRNA, serum-starved and either nonstimulated or stimulated with 100 nM insulin for 30 minutes. The fixed cells were stained for F-actin and morphological changes in the actin cytoskeleton (appearance of the `insulin-responsive' actin phenotype) were quantified as described in the Materials and Methods. (C) Re-expression of annexin A2 in annexin-A2-depleted cells restores the ability to insulin-dependently remodel F-actin. Western blotting of total cell lysates shows comparable levels of annexin A2-GFP expression in annexin A2 siRNA and mock-treated cells. GFP-expressing cells remained unable to undergo insulin-induced F-actin changes, whereas cells re-expressing annexin A2-GFP responded similar to nondepleted cells and displayed the insulin-induced actin phenotype. ^**P<0.0005.