Cells were seeded in a six-well plate (5 × 105 H9c2 cells/well) cultured in high-glucose DMEM supplemented with 10% FBS and then treated with CoCl2 or HT for indicated doses and time. Proteins were isolated by lysis buffer (Beyotime).
After the animals were sacrificed, the heart tissues were disrupted by homogenization on ice with lysis buffer. After centrifugation, protein extracts were collected.
Protein lysates were separated on 10% SDS-PAGE, transferred onto the PVDF membranes, and blocked with 5% non-fat milk for one hour, and then incubated with primary antibodies overnight at 4 °C and secondary antibodies for one hour at room temperature. Membranes were again washed with TBST and immunoreactive proteins were visualized using ECL Western blotting detection reagents (Cell Signaling Technology) were used to detect immunoreactive proteins[17 (link)].
After the animals were sacrificed, the heart tissues were disrupted by homogenization on ice with lysis buffer. After centrifugation, protein extracts were collected.
Protein lysates were separated on 10% SDS-PAGE, transferred onto the PVDF membranes, and blocked with 5% non-fat milk for one hour, and then incubated with primary antibodies overnight at 4 °C and secondary antibodies for one hour at room temperature. Membranes were again washed with TBST and immunoreactive proteins were visualized using ECL Western blotting detection reagents (Cell Signaling Technology) were used to detect immunoreactive proteins[17 (link)].
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