FIGURE 3

Cdc42 is activated by VEGF and is upstream of SAPK2/p38 in VEGF/VEGFR2 signaling. (a) Quiescent HUVEC were treated for various periods of time with 5 ng/ml VEGF. They were then extracted and incubated with GST-CRIB to adsorb activated Cdc42. The samples were then loaded on 12% SDS–PAGE, transferred onto a nitrocellulose membrane and blotted for Cdc42. (b) Using the CaCl₂ transfection method, NIH 3T3 cells were transfected with either pAlter-VEGFR2, pCDNA3-myc-Cdc42 V12 or pCDNA3-myc-Cdc42 N17. At 48 h after transfection, quiescent cells were left untreated or exposed to 10 ng/ml VEGF for 1 min. Cells were then processed for Cdc42 assay as in (a). (c) NIH 3T3 cells were transfected with pAlterMax/VEGFR2 and pCDNA3 and selected for stable expression using 400 g/ml G418. Different clones were obtained and cells from these clones were grown in selective media, plated and extracted in Laemli sample buffer. A total of 35 g of proteins were loaded on 8.5% SDS–PAGE, transferred onto nitrocellulose and analysed for VEGFR2 expression. The clone 4F2 expresses the highest amount of VEGFR2 and was selected for further experiments. In (d) 4F2 cells were plated for 24 h. Then, using the CaCl₂ method, they were cotransfected with pCDNA3-HA-p38, pSVT7-LT-MAPKAP K2 and pCINeo (empty vector), or pCDNA3-myc-Cdc42 V12 or pCDNA3-myc-Cdc42 N17. After 48 h, cells were left untreated or exposed to 10 ng/ml VEGF for 10 min. Cells were extracted and LT-MAPKAP kinase-2 was immunoprecipitated using a KT3 mouse antibody. Then LT-MAPKAP K2 was assayed in the presence of [-³²P]ATP using recombinant HSP27 as a substrate. Proteins were separated by SDS–PAGE and the kinase activity was visualized using a PhosphorImager system by measuring ³²P incorporation into the specific substrate. Results are expressed as the ratio of kinase activity of stimulated cells over unstimulated cells. Representative results from two distinct experiments are shown. In (e), 4F2 cells were cotransfected with pCDNA3-HA-p38 and pCDNA3 (empty vector) or pCDNA3-myc-Cdc42 N17. After 48 h, quiescent cells were left untreated or exposed to 10 ng/ml VEGF for 10 min. Cells were extracted and HA-p38 was immunoprecipitated using the HA-tag antibody. The immunocomplexes were loaded on 10% SDS–PAGE, transferred onto nitrocellulose and blotted with phospho-p38 antibody. The membrane was stripped and reprobed with HA-tag to ensure equal protein loading