Enhanced chemi luminescence (ecl)

The cells in the high-speed growth phase were treated with a 4-fold dilution of the Mg alloy extract and medium for 24 h, washed with PBS three times and added RIPA cell lysate containing 1% protease inhibitor and 1% phosphatase inhibitor. A cell scraper was used to hang the adherent cells. The lysate was collected, vortexed and placed on ice for 30 min to be fully lysed. After 12 000 g refrigerated centrifugation for 10 min, the supernatant was transferred to a clean EP tube. The Micro BCA™ Protein Assay Kit (Thermo Scientific, USA) was used to measure the protein concentration of the samples, and then a 5× loading buffer was added. The samples were heated at 100°C for 15 min to denature the proteins. The proteins were separated by using 10% SDS-PAGE, and the same amount of total protein (20 μg) was added to the loading well. After the electrophoresis, the proteins were transferred to the PVDF membrane, which was blocked with 5% skim milk for 2 h, incubated with the primary antibody (Table 2) at 4°C overnight, then washed 3 times for 5 min with TBST buffer. The secondary antibody was used to incubate for 2 h at room temperature. After washing with TBST three times, the targeted proteins were visualized with enhanced chemiluminescence (ECL, Beyotime Biotechnology Co., China) in the Molecular Image ChemiDoc XRS⁺ system (Bio-Rad Laboratories Inc., USA).

The RIPA-lysed ground mouse myocardial tissue was treated with centrifugalization (12,000 rpm, 15 min) to collect the supernatant. Protein concentration was measured with the use of the BCA protein quantification kit. Equal amount of 10 μL protein samples were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (230 mA, 2.5 h) that was subjected to 1 h of cultivation with 5% bovine serum albumin solution. The PVDF membrane was then cut out according to the different molecular weights of the protein and placed in the antibody-incubation box. The diluted corresponding Ι antibodies were used for overnight incubation (4°C). After incubation, the corresponding II antibody was placed into the box for 2 h of indoor cultivation. ECL (Beyotime, Shanghai, China) was employed for the identification of the expression of the corresponding protein bands in the bioimager and ImageJ software (Image J Software, Bethesda, USA) for the gray value analysis of the protein band, with GAPDH as an internal reference.