Nbt bcip system

The hearts were homogenized in lysis buffer containing (mmol/l) 150 NaCl, 50 Tris-HCl, 5 EDTA.2Na, and 1 MgCl₂ plus protease inhibitor (Sigma Fast; Sigma, USA). The protein concentration was determined by the Lowry method, [22] (link) and bovine serum albumin (BSA) was used as the standard. Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes incubated with mouse monoclonal antibodies for catalase (CAT; 1∶2000; Sigma, USA), rabbit polyclonal antibodies for superoxide dismutase (SOD-2; 1∶1000; Sigma, USA) and Gp91phox (1∶1000; BD, New Jersey, EUA) and rabbit polyclonal antibodies for AT₁ (1∶500; Santa Cruz Biotechnology, CA, USA) and GAPDH (1∶1000; Santa Cruz Biotechnology, CA, USA). After washing, the membranes were incubated with either an alkaline phosphatase conjugated anti-mouse IgG (1∶3000, Abcam Inc., Cambridge, MA, USA) or an anti-rabbit antibody (1∶7000; Santa Cruz Biotechnology, CA, USA). The bands were visualized using a NBT/BCIP system (Invitrogen Corporation, CA, USA) and quantified using ImageJ software (National Institute of Health, NIH). The results were calculated using the ratio of the density of specific proteins to the corresponding GAPDH.

Coronary samples were homogenized in a lysis buffer containing 150 mM NaCl, 50 mM
Tris-HCl, 5 mM Na₂EDTA, and 1 mM MgCl₂, plus a protease
inhibitor (Sigma Fast; Sigma). Protein concentration was determined by the Lowry
method (27 ) and bovine serum albumin (BSA) was
used as the standard. The same amount of proteins (50 µg) were denatured and
separated by SDS-PAGE (10%) and transferred onto a PVDF membrane (Millipore,
Germany). Membranes were blocked with 5% BSA at room temperature in a TBS buffer plus
Tween 20 (0.1%) before incubation with polyclonal anti-mouse antibody for
Mn-superoxide dismutase 2 (SOD-2; 1:1000-Sigma), monoclonal anti-mouse antibody for
catalase (1:2000-Sigma), and polyclonal anti-mouse antibody for β-actin
(1:1500-Sigma). After washing, the membranes were incubated with an alkaline
phosphatase conjugated anti-mouse IgG (1:3000, Abcam Inc., USA). The bands were
visualized using an NBT/BCIP system (Invitrogen Corporation, USA) and quantified
using ImageJ software (National Institute of Health, USA).