Anti-P-Tyr and anti-PLCβ1monoclonal antibodies were purchased from Biosource (Prodotti Gianni, Milano, Italy) and Upstate (Lake Placid, NY, USA), respectively while rabbit anti-SHP-2 (C-18) polyclonal antibody was from Santa Cruz Biotechnology (CA, USA). Protease inhibitor cocktail was obtained from Roche Diagnostic (Indianapolis, IN, USA). Anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) were from (Calbiochem (Darmstadt, Germany).
Skin fibroblasts from 6 healthy subjects from the staff of the Department of Clinical and Experimental Medicine at the University of Padova, who gave their informed consent, were obtained via biopsy and individually cultured in F-10 HAM medium with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 4 mmol/l glutamine, as previously described [19, 27, 28] and used after the third passage. To assess the effects of Ang II, cells were incubated with 100 nM Ang II for 1 hour. This concentration was chosen, since it was clearly seen to induce Ang II signaling in previous reports [19, 29, 30]. To assess the effects of phosphatase activity on protein phosphorylation, cells were incubated with 1 mM vanadate overnight. To examine the effect of Ang II type 1 receptor signaling blockade, cells were preincubated for 30 min with 100 μM losartan and then treated as described above. This concentration was also chosen based on a previous report .
Anti-SHP-2 and Anti PLCβ1 immunoprecipitation was done using confluent cells. These were scraped, washed in buffer and extracted (1 h at 4°C with buffer C (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Nonidet-P-40, 1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, protease inhibitor cocktail). After centrifugation, 200 μg of supernatant protein were diluted 1:1 in 20 mM Tris-HCl, pH 7.5, containing 1 mM sodium orthovanadate and protease inhibitor cocktail, precleared with protein A-Sepharose, and anti-SHP-2 or anti PLCβ1antibodies bound to protein A-Sepharose were added at 4°C. This was then incubated overnight, immunoprecipitates were washed 3× in buffer D (25 mM imidazole, pH 7.0, 1 mM EDTA, 0.02% NaN3, 10% glycerol, 10 mM B-mercaptoethanol, 10 mg/ml leupeptin, 50 mM PMSF), resuspended and then submitted to gel electrophoresis (SDS-PAGE; 8% or 10% gels), transferred by blotting to nitrocellulose membranes and immunostained with the appropriate antibodies/second antibodies.
Phosphatase activity was measured at 30°C using nitrophenyl phosphate (pNPP) (10 mM pNPP as substrate in 100 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and PTPase immunoprecipitates from 200 mg cell-protein content in Buffer D). After 10 min at 30°C, the reaction was quenched with 950 μl of 1 M NaOH. Absorbance at 405 nm was measured and in all cases the substrate-to-product conversion was less than 5%. All the reagents were from Sigma (Milano, Italy). Results are expressed as nanomoles per minute per 200 mg cell protein immunoprecipitate.
RGS-2 gene silencing was done using chemically synthesized siRNA that mapped to exon 5 of RGS-2 gene (Silencer Pre-Designed siRNA, Ambion, Austin, USA) as previously described . Fibroblasts (2 × 105 cells) were plated the day before transfection in 6-well plates in growth medium without antibiotics containing 10% FBS. On the day of transfection, siRNA was incubated with Lipofectamine 2000 diluted in OPTI-MEM I (Invitrogen, Carlsbad, USA) following manufacturer's instructions (Invitrogen, Carlsbad, USA). We have chosen for our experimental protocol RGS-2 siRNA mapping for exon 5 at a concentration of 50 nmol/l and transfected the oligos with Lipofectamine 2000 (4 mg/ml) as previously reported . Following 20 min incubation at room temperature, the obtained complexes were added drop-wise onto the cells subcultured in replaced cell-culture medium. The cells were maintained in a 37°C incubator until analysis. The medium was changed to medium with no siRNA 12 h after transfection. Fluorescein-conjugated siRNA (Control (non-silencing), Fluorescein, Qiagen, Hilden, Germany) with no sequence identity for any human gene was used as negative control to exclude non-specific effects and to monitor the efficiency of transfection while GAPDH siRNA was used as positive control (Ambion, Austin, TX USA). Silencing was assessed by western blot and found to be 44% as previously reported . Horseradish peroxidase (HRP)-conjugated (Amersham Pharmacia, Uppsala, Sweden) antibody was used as secondary antibody and visualized with chemiluminescence, which was captured on radiograph film. Exposed films were digitized by scanning densitometry and protein levels were calculated using National Institutes of Health (NIH) Image software (NIH, Bethesda, Maryland, USA). β actin was used as housekeeping gene and the ratios between RGS-2 and β actin western blot products were used as index of RGS-2 protein expression and expressed as densitometric arbitrary units.
Data were evaluated statistically as normally distributed continuous variables and comparisons were performed using one-way ANOVA (Statistica, Statsoft Inc, Oklahoma City, OK, USA). Results with p < 0.05 were considered significant and data values are presented as mean±SD.