For Western blot (WB) analyses 50 µg proteins/sample were used either from cell culture (naive MSCs and after 7, 14, and 28 days after MiCi-HIF1α transfection) or from myocardial samples. Samples were loaded and separated on 7.5% acrylamide gels. After electrophoresis, proteins were blotted on PVDF membrane (#162–0261; Bio-Rad Laboratories, Hercules, California, U.S.) and stained with internal control ß-actin (Sigma-Aldrich, A2066, dilution 1/2000) and anti-HIF1α antibody (Abcam #51608, dilution 1/1000). As secondary antibody, goat anti-rabbit IgG (Abcam #6721, dilution 1/2000) was used. As a marker, a pre-stained Pageruler Standard was used (Thermo Scientific #26616). The blots were incubated with a luminol detection reagent (Thermo Fisher) and visualised on a Chemi-Smart system (Vilber Lourmat, Collégien, France) with optimised exposure times.
Chemi smart system
Manufactured by Vilber
Sourced in France
The Chemi-Smart system is a high-performance chemiluminescence and fluorescence imaging platform designed for a variety of applications in life science research. The system features a sensitive camera, advanced optics, and a temperature-controlled imaging chamber to capture and analyze chemiluminescent and fluorescent signals.
Automatically generated - may contain errors
2 protocols using chemi smart system
1
Quantifying HIF1α in Stem Cells and Myocardium
2
Immunoblotting Analysis of Liver Proteins
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For immunoblotting analysis, total cell lysates were first prepared from mouse livers and primary hepatocytes using lysis buffer containing 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, and protease inhibitor cocktail (Cat. No. 4693132001; Roche). Lysate proteins (50 µg) were separated by SDS-PAGE and transferred onto a PVDF membrane. After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich). The blots were then incubated with HRP-conjugated secondary antibodies and visualized using ECL substrate and the Chemi-Smart system (Vilber Lourmat, France). The intensities of the protein bands were determined using ImageJ software (NIH, USA).
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