1 step nbt bcip reagent

Total protein from cultured cells were lysed in RIPA buffer with protease inhibitor (Beyotime, Shanghai, China). The protein was quantified using a BCA assay kit (Beyotime, Shanghai, China). A total of 20 μg of total protein were separated by 10% SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and then reacted with primary antibodies against METLL3, METLL14, FTO, ALKBH5 and β-actin (all from Abcam, Cambridge, UK). After being extensively washed with PBS containing 0.1 % Triton X-100, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody for 30 min at room temperature. The bands were visualized using 1-step TM NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, USA) and detected by an Alpha Imager (Alpha Innotech, San Leandro, CA, USA).

About 20 μg of total protein form heart tissues was extracted and separated by 10 % SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and then reacted with primary antibodies against Anti-IL-27RA (1:500) and β-actin (1:1000) (all from R&D Systems, Minneapolis, MN, USA). After being extensively washed with PBS containing 0.1 % Triton X-100, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody for 30 min at room temperature. The bands were visualized using 1-step TM NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, USA) and detected by an Alpha Imager (Alpha Innotech, San Leandro, CA, USA).

Protein lysates were obtained from 1 × 10⁶ cultured cells with RIPA buffer and boiled at 100°C for 10 min. Approximately 20 μg protein lysates were extracted from each sample, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After being blocked in 5% nonfat milk at room temperature for 1 h, the interested protein was probed with primary antibody against human PRDM16 (1:1000 dilution, Abcam), GAPDH (1:1000, Proteintech), Vimentin (1:1000, Proteintech), E-cadherin (1:1000, Proteintech), N-cadherin (1:1000, Proteintech), MMP3 (1:1000, Proteintech), or PC (1:1000, Proteintech) at 4°C overnight and then incubated with goat antirabbit IgG or goat antimouse IgG (1:5000 dilution for both; Proteintech) at room temperature for 1 h and detected with enhanced chemiluminescence reagents (Thermo Fisher Scientific, Shanghai, China). The bands were visualized using 1-step^TM NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, United States) and detected by the Alpha Imager (Alpha Innotech, San Leandro, CA, United States). The protein expression level was quantified and normalized to GAPDH protein expression by densitometry using Image-J. Statistical data was obtained from three independent experiments.