AP39 through AMPK-ULK1-FUNDC1 pathway regulates mitophagy inhibits pyroptosis improves doxorubicin-induced myocardial fibrosis
Description
Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Prof. Dr. Jun Yang(1996020012@usc.edu.cn).
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Myocardial tissue or cellular proteins were extracted using a protease inhibitor-containing cell lysate. Protein quantification was performed using a BCA quantification kit (Beyotime, Shanghai, China, P0010S). After equalization of protein concentrations, an appropriate amount of protein loading buffer was added, and the proteins were denatured by heating at 95 degrees for 10 min and separated by 10–12% SDS-PAGE. The proteins were wet transferred to PVDF membranes and blocked with a 5% fat-free milk-blocking solution for 1 h. CSE (12217-1-AP), α-SMA (14395-1-AP), collagen III (22734-1-AP), MMP8 (17874-1-AP), MMP13 (18165-1-AP), TIMP1 (6644- 1-AP), caspase1 (22915-1-AP), IL-1β (16806-1-AP), beclin1 (11306-1-AP), LC3 (18725-1-AP), P62 (18420-1-AP), AMPK (10929-2-AP), ULK1 (20986-1-AP), and GAPDH (60004-1-IG) were purchased from Proteintech (Chicago, USA). p-AMPK (2535), GSDMD (39754), and NLRP3 (15101) were purchased from CST (Cell Signaling Technology, Boston, MA, USA). p-ULK1 (ab133747) primary antibody was purchased from Abcam (Cambridge, UK). FUNDC1 (PA5-75706) primary antibody was purchased from Thermofish (Thermo Scientific, Massachusetts, USA). Mouse (SA00001-1)/rabbit(SA00001-2) secondary antibodies were purchased from Proteintech (Chicago, US). The primary antibody was incubated overnight at 4 degrees, then washed 10 min × 3 times with TBST. The secondary antibodies of the related species were incubated for 1 h at room temperature, washed again for 10 min × 3 times with TBST, and photographed using ECL solution. The images were obtained using the BIO-RAD XRS+ imaging system (BIO-RAD Life Science, California, USA). The comparative data were analyzed using ImageJ software (NIH, Bethesda, USA), and GAPDH was used as an internal reference to calculate the relative values of each protein.