Figure 4

DUSP3 depletion affects PKC activation but is dispensable for ERK1/2, JNK and EGFR in HUVEC cells. HUVECs were transfected with non-targeting siRNA (siCTL) or with DUSP3 targeting siRNA (siDUSP3-1 and siDUSP3-2). 24 h before stimulation, cells were washed and let to rest overnight in 2% serum containing medium. Cells were then activated with b-FGF (10 ng/mL) for the indicated time points theb lysed. Cell lysates were resolved on SDS-PAGE and immuno-reacted with (Ai) anti-phospho-ERK1/2 (Thr202/Tyr204) and ERK as an internal loading control, (Aii) anti-DUSP3 and anti-GAPDH (Aiii) Quantification of the phosphorylation levels or ERK was determined by densitometric analysis and is shown as a ratio of pERK/ERK. Results are presented as mean ± SEM and are representative of 3 independent experiments. (B) SAPK/JNK kinase assay. JNK was immunoprecipitated from siCTL and siDUSP3 transfected cell lysates. After transfer of the JNK immunoprecipitates, nitrocellulose membranes were immuno-reacted with anti-phospho-c-Jun and anti-c-Jun antibodies (Bi). Quantification of the phosphorylation levels or JNK substrate, c-Jun, was determined by densitometric analysis and is shown as a ratio of p-c-Jun/c-Jun (Bii). (C) EGFR phosphorylation. EGFR was immunoprecipitated from non-stimulated and EGF (100 ng/ml) stimulated HUVECs transfected with siCTL or with siDUSP3. Immunoprecipitates were immunoreacted with anti-phosphotyrosine antibody 4G10. Membranes were stripped and re-bloted with anti-EGFR antibody. (D) Western blot for p-Akt and Akt on cell lysate from FGF stimulated siCTL and siDUSP3 transfected conditions. (Ei) Western blot for phospho-PKC (Ser660) on cell lysates from FGF stimulated cells in the conditions indicated. ERK was used as a loading control. (Eii) Quantification of the phosphorylation levels or PKC was determined by densitometric analysis and is shown as a ratio of phospho-PKC/ERK. Results are presented as mean ± SEM and are representative of 4 independent experiments. *p < 0,05; **p < 0.01; ***p < 0,001.