3-Methyladenine (3-MA)

Protocol tips

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	After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA).

Protocol tips

After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA).

Publication protocol

After treatments, MAEC and HL-1 cells were washed three times with cold PBS and lysed in RIPA buffer (Beyotime, Shanghai, China) containing the Protease Inhibitor Cocktail and protease inhibitor PMSF for 30 min. Lysates were centrifuged at 12000rpm for 5 min at 4 °C. The protein samples were boiled in SDS-loading buffer for 10 min and were separated by 10% SDS-PAGE. Immunoblotting was performed with primary antibodies against parkin (1: 500), LC3 (1: 1000), P62 (1: 1000), ERRα (1: 1000), eNOS (1: 200), p-eNOS (1: 200), TOM20 (1: 1000), cleaved-caspase-3 (1: 1000), and GAPDH (1: 1000) followed by the incubation with HRP-labeled secondary antibody. Protein bands were visualized using ECL Plus detection reagents with Amersham ECL Detection Systems (Amersham, Buckinghamshire, UK). Densitometric analysis was performed using Image Lab Software (Bio-Rad, USA).

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Manufacturer protocol

Download the product protocol from Selleckchem.com for 3-Methyladenine (3-MA) below.

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