Regulation of COX-2–mediated signaling by α3(IV)NC1 in hypoxic ECs. (A) Autoradiogram showing expression of COX-2 mRNA on α3(IV)NC1 treatment. Celecoxib was used as a positive control. (B) Western immunoblot of MLEC extracts using antibodies specific to COX-2. (C) β3 integrin-null MLEC extracts were analyzed by Western blotting using antibodies specific to COX-2. (D) MLECs treated with and without α3(IV)NC1 were exposed to hypoxia for approximately 12 hours and stained with COX-2 antibody. Images were viewed using a Zeiss AX10 microscope (Carl Zeiss) with a 40×/0.75 NA objective lens, captured using a Flex Digital IEEE-1394 camera (Sheerin Scientific), and processed using SPOT advanced imaging software version 3.1.0 (Diagnostic Instruments) and Adobe Photoshop 7.0 (Adobe). (E) Autoradiogram showing expression of bFGF mRNA on α3(IV)NC1 treatment in cow pulmonary artery ECs. The top half of the autoradiogram (0.475 bFGF probe) shows inhibition of expression of bFGF mRNA on treatment with α3(IV)NC1 or IFN-α (positive control). (F,G) Similar to panel B, cow pulmonary artery ECs and MLEC extracts were immunoblotted with antibodies specific for bFGF and VEGF. (H) α3 integrin-null ECs were treated with α3(IV)NC1 and exposed to hypoxic conditions and cell extracts were immunoblotted with COX-2 antibody. 18S RNA (A,E) and actin (B,C,F-H) levels were shown as loading controls.