Quantity one image analyzer software

Heart and aorta were removed 8 weeks after electroacupuncture stimulation. The tissues were lysed in RIPA buffer containing phosphatase and protease inhibitors and the protein content was quantified with a Bio-Rad kit (Bio-Rad, Hercules, CA, USA) following the conditions suggested by the manufacturer. Equal amount of total protein was subjected to SDS-PAGE and blotted. The PVDF Membranes (Millipore, Billerica, MA, USA) were incubated with specific primary antibodies overnight at 4°C followed by HRP-labeled secondary antibodies for 1 h at room temperature. The primary antibodies used included AT1R antibody (1:300), ETAR antibody (1:300), eNOS antibody (1:1000) and iNOS anibody (1:1000) (Abcam, Cambridge, MA, USA). The targeted proteins were detected by using enhanced chemiluminescence system (Pierce,Rockford,IL, USA). Densitometric analyses of Western blots were performed using the Quantity One image analyzer software (Bio-Rad, Hercules, CA, USA). The analysis was performed using volume rectangular tool. For background subtraction the same area was selected above and below the bands using the global method for background subtraction [19 (link)].

Bands from immunoblots or agarose gels were quantified by using Quantity One image-analyzer software (Bio-Rad Laboratories). All culture assays were normalized on the basis of cell viability by using the CellTiter-Glo assay from Promega. Each experiment was performed at least in triplicate and data were expressed as mean ± SEM. Comparisons between two or four samples were performed using Student’s t-test or one way analysis of variance (47 (link)) with post hoc Tukey’s test (Prism software, GraphPad Inc, San Diego, CA).

To determine the levels of phosphorylation of CaMK II-δ and the expression of Na_v 1.5, 7 groups of heart tissue were properly used and analyzed after perfusion with either K-H solution in absence (control) or presence of Bay K 8644 (200 nM), ATX-II(3 nM), ATX-II (3 nM) + Bay K 8644 (200 nM), Bay K 8644 (200 nM) + TTX (1 μM), ATX-II (3 nM) + TTX (1 μM), ATX-II (3 nM) + Bay K 8644 (200 nM) + TTX (1 μM). Total protein of left ventricular myocardium was extracted, immunoprecipitation and Western blotting analysis were performed using a standard method. In brief, tissue lysates were first incubated with magnetic beads covalently conjugated with anti-phospho-Akt substrate (RXXS*/T*) rabbit mAb (Cell Signaling Technology, #8050) and anti-phospho-PKA substrate antibody (RRXS*/T*) Ab (Cell Signaling Technology, #9621) at 4 °C overnight, respectively. Sample buffer mixed with the washed beads was heated at 95 °C for 5 min and subjected to SDS-PAGE. Then the proteins were blotted with rabbit anti- CaMK II-δ Ab (Abcam, ab90445), and anti-Na_v 1.5 Ab (Abcam, ab56240), respectively. Blotted antibodies were visualized by HRP-conjugated mouse anti-rabbit IgG mAb (Cell Signaling Technology, #5127) and ECL detection system (Millipore). Densitometric analyses of blots were performed using the Quantity One image analyzer software (Bio-Rad, Richmond CA, USA).