ETAPAS QUE DESARROLLAN LOS CÍRCULOS DE CALIDAD

RIPA buffer (lysis buffer) :

NaCl 150 mmol/L Tris-HCl 50 mmol/L Nonidet P40 1.00% (m/v) Deoxycholat 0.25% (m/v) SDS 0.10% (m/v) + Na3VO4 1 mmol/L + PMSF 1 mmol/L added

+ Complete Protease inhibitor, 25x stock solution freshly

NaF 50 mmol/L before

Na4P2O7 50 mmol/L use SDS sample buffer (5x): Tris-HCl (pH 6.8) 3.125 mol/L 2 mL Glycerol 10 mL (50% (v/v)) SDS 20% (m/v) 5 mL DTT 16% (m/v) 2.5 mL Pyronin Y 5% (m/v) 0.1 mL H2O ad 20 mL Resolving gel 10%:

Acrylamide 30%-bisacrylamide 0.8% solution (Roth) 5.0 mL

1.5 mol/L Tris-base pH 8.8 (Roth) 3.75 mL

10% SDS 0.15 mL

2 MATERIALS AND METHODS

The solution was degassed for 10 min, since polymerization requires anaerobic conditions. Afterwards, the polymerization reaction was started by addition of:

15 µl TEMED (cross linker)

75 µl APS 10% (m/v) (radical starter)

Stacking gel:

Acrylamide 30%-Bisacrylamide 0.8% solution (Roth) 1.7 mL

1.25 mol/L Tris-base pH 6.8 1 mL

10% SDS 0.1 mL

H2O 7 mL

The solution was degassed for 10 min. Afterwards, the polymerization reaction was started by addition of:

20 µl TEMED (cross linker)

100 µl APS 10% (m/v) (radical starter) Electrophoresis buffer: Tris base 3 g Glycin 14.4 g SDS 1 g H2O dest. ad 1,000 mL Anode buffer I pH 10.4: Tris base 30 g Methanol 200 mL H2O dest. 800 mL Anode buffer II pH 10.4: Tris base 3 g Methanol 200 mL H2O dest. 800 mL

2 MATERIALS AND METHODS Cathode buffer pH 7.6:

ε-Amino-n-caproic acid 5.2 g

Methanol 200 mL

H2O 800 mL

Tris buffered saline solution containing Tween, pH 8.0 (TBS-T): Tris base 3 g (0.02 mol/L)

NaCl 11.1 g (0.2 mol/L) Tween 20 1 mL (0.1 %) H2O dest. ad 1,000 mL

Primary antibodies:

Anti-HSP32 (HO-1) monoclonal mouse anti-human antibody Anti-HSP70 polyclonal goat anti-human antibody

Anti-phospho-p38 MAPK polyclonal rabbit anti-human antibody Anti-phospho-ERK monoclonal mouse anti-human antibody Anti-phospho-JNK/SAPK monoclonal mouse anti-human antibody Anti-α-tubulin rabbit antibody

Secondary antibodies:

Peroxidase-conjugated goat anti-rabbit IgG Peroxidase-conjugated goat anti-mouse IgG Peroxidase-conjugated donkey anti-goat IgG

2.4.2 Sample preparation

For detection of proteins, whole cell lysates of HUVEC were prepared. HUVEC were cultured in 12-well plates until confluence and were either left untreated or stimulated with ANP (10-9 – 10-6 mol/L), 8-Br-cGMP (10-3 mol/L) or cANF (10-6 mol/L). After the indicated times, the medium was removed, cells were washed three times with ice cold PBS and lysed by addition of 100 µl RIPA buffer. In some cases, Western blots were performed after pretreatment of HUVEC with U0126 (50 µmol/L), PD 98059 (50 µmol/L), or SP 600125 (10

2 MATERIALS AND METHODS

µmol/L) for 1 h. After addition of RIPA buffer, the cells were scraped off the plates with a cell scraper, the lysate was transferred into reaction tubes (Eppendorf), and sonicated (Sonoplus, Bandelin, Germany) for approx. 5 sec. The homogenized samples were clarified by centrifugation at 21,910 x g for 10 min at 4°C and the resulting supernatants were collected and subsequently quantified for protein according to the method of Lowry (Lowry 1951). 5x sample buffer was added to the remaining probes and the samples were heated to 95°C for 5 min to achieve protein denaturation. Afterwards, the samples were either subjected to SDS/PAGE or stored at -20°C until further analysis.

2.4.3 SDS polyacrylamide electrophoresis

The separation of proteins was carried out by denaturing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (Laemmli 1970). The separation of proteins solely according to their size is achieved by addition of SDS which complexes proteins in a constant weight ratio and masks their charge by its own strongly negative charge resulting in identical charge densities on the surface. Disulphide bridges are cleaved by reducing agents (DTT). The molecular weight of the separated proteins was determined by comparison with a molecular weight standard (biotinylated protein marker, Cell Signaling, 6.5 kDa – 165 kDa). The electrophoresis was carried out with a BioRad Mini Protean II Cell (BioRad, München, Germany).

2.4.4 Semidry blotting

Using a trans-blot semidry system (BioRad), the separated proteins were electrophoretically transferred to a polyvinylidenfluoride membrane (Immobilon-P, 0.45 µm pore size, Amersham) which was incubated for 5 min in methanol, for 5 min in water, and stored in anode buffer II before usage. Six sheets of blotting paper were soaked with anode buffer I and three sheets with anode buffer II and rolled onto the blotter without bubbles. The membrane and the gels were added and covered with nine sheets of blotting paper moistened with cathode buffer. Blotting was carried out for 1 h at 64 mA. Afterwards, the membrane was dried at 80°C for appox. 30 min. Unspecific binding sites were saturated by shaking the membrane in 5% (m/v) Blotto (BioRad) in TBS-T.

2 MATERIALS AND METHODS

2.4.5 Incubation with antibodies

Antibody solutions were prepared 1:1,000 or 1:10,000 in 1% Blotto in TBS-T. The blots were incubated with the first antibody overnight at 4°C under constant shaking. Following three washes with TBS-T, the second horseraddish peroxidase-conjugated antibody was added and incubated for at least 1 h at room temperature. After another three washes with TBS-T, the protein bands were visualized by a chemoluminescence reaction using an enhanced chemoluminescence protein detection kit (NEN, Cologne, Germany), containing the enzyme substrate luminol and chemical enhancers, and a Kodak Image station (Kodak digital science, Stuttgart, Germany). Luminol is oxidized in the presence of H2O2 and peroxidase, releasing

light (428 nm) which is aquired by the Kodak Image station.

figure 7: principle of the chemoluminescence reaction

2.5 cGMP measurement