p38 and Akt pathways are differentially induced by soluble and matrix-bound VEGF₁₆₅. (A) Confluent HUVEC cultures were incubated in the presence or absence of soluble (Vs) or matrix-bound VEGF₁₆₅ (Vb; 200 ng/ml) for 5 min. Cell lysates were obtained, resolved by SDS-PAGE, and probed with antibody against phospho-p38 (43 kD). The blot was then stripped and reprobed with total p38 antibodies for normalization. (B) HUVECs were exposed to Vs or Vb (200 ng/ml) at the indicated times. The blot was then stripped and reprobed with total p38 antibody for normalization. (C) Same settings as B, but membrane was immunoblotted with phospho-Akt (S473) and total Akt (60 kD). Densitometry was determined by phospho-p38 over total p38 (n = 6) (D) or phospho-Akt over total Akt (n = 6) (E).