To determine the amount of CAV3 in the hearts of sham (n = 4), SSI (n = 6), SH+VP (n = 4), and SSI+VP (n = 6) mice, homogenates of left ventricles were submitted to immunoblotting 6, 12, and 24 hours after the CLP or sham procedure. Hearts of mice were homogenized in the modified RIPA buffer lysis (Tris HCl 0.05°M (pH°7.4); NaCl 0.15°M; EDTA 0.001°M (pH°8.0); SDS 0.1%) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and the phosphatase inhibitors (Na3VO4 0.001°M; NaF 0.025°M; Na4P2O7 0.0005°M). This buffer does not separate cytosolic protein from plasma membrane protein. Equal concentrations (50°μg/well) of total proteins (homogenate) were resolved on 10% SDS-Page gels and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Amersham, UK). The membranes were blocked with 5% albumin for two hours and incubated overnight at 4°C with the primary antibodies: anti-caveolin-3 (mouse monoclonal antibody, 1 : 10000; BD Transduction Laboratories) and anti-GAPDH (rabbit monoclonal antibody, 1 : 1000; Cell Signaling Technology). Then, the blots were washed and incubated with HRP-conjugated secondary antibodies for one hour at room temperature. Membranes were washed, developed using ECL (Amersham Pharmacia Biotech), and viewed with ChemiDoc XRS (BioRad). Image analysis was performed using the public domain ImageJ program (developed at the National Institutes of Health and available at http://rbs.info.nih.gov/nih-image/) with the “Gel Analysis” function. Analysis results are represented by the values of each band; each value is proportional to the integrated density value (IDV) of the specific band, which corresponds to the arbitrary unit (AU). GAPDH was used to determine equivalent loading conditions.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。