Ne pertm nuclear and cytoplasmic extraction kit

The nuclear and cytoplasmic lysates were extracted using a NE-PERTM nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific, Inc.). After modified with biotin, a total of 100 pmol wild-type or mutated miR-196a oligonucleotides were added to the lysates for 12 h at 4°C. Then, agarose beads (Invitrogen; Thermo Fisher Scientific, Inc.) were added into the lysates. After incubation at 4°C for 4 h, the proteins were extracted and analyzed by western blotting. Poly (G) (5′-GGGGGGGGGGGGGGGGGGGGG-3′) was labeled with biotin and used as a negative control.

Total cytoplasmic and nuclear proteins from OVCAR-3 cells were extracted with RIPA buffer (Thermo Fisher, USA). NE-PERTM Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher, USA) was added with protease and phosphatase inhibitor cocktail (Thermo Fisher, USA). BCA protein assay kit (Beyotime Biotechnology, China) was used for quantification. Protein (30 μg) was loaded onto SDS-PAGE gel and transferred to a Hybond-P PVDF membrane (GE Healthcare, USA). 5% fat-free milk in TBST (Tris-buffered saline with 0.1% Tween 20) was used for blocking. The following antibodies were incubated with the membranes at 4°C overnight: mouse anti-E-cadherin (1 : 2000, CST: 3195, USA), rabbit anti-vimentin (1 : 2000, CST: 5741, USA), rabbit anti-Snail (1 : 1000, Abcam: ab216347, USA), and actin (1 : 5000, Abcam: ab8227, USA) as an internal control. Protein expression was visualized on X-ray films using the HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1 : 5000, Thermo Fisher, USA) and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, USA). Band intensities were quantitated using Image Pro Plus 6.0 software. The results were presented as the density ratio of the target protein band to the internal control.

Subcellular fractions were prepared from groups of five 10–15-week-old and 30-50-week-old wt and cTnI-G203S hearts, an NE-PER^TM Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific, 78833), supplemented with cOmplete^™, Mini, EDTA-free Protease (Roche, 4693159001) and PhosStop^TM phosphatase (Roche, 4906837001) inhibitor tablets, according to manufacturer protocols. Blots were probed with the following primary antibodies: Total mTOR, mTOR (7C10) rabbit mAb (Cell Signaling Technology, 2983, 1:1000); Active mTOR, phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (Cell Signaling Technology, 5536, 1:1000); Total SRP6, S6 ribosomal protein (5G10) rabbit mAb (Cell Signaling Technology, 2217, 1:1000); Active SRP6, phospho-S6 ribosomal protein (Ser235/236) (2F9) rabbit mAb (Cell Signaling Technology, 4856, 1:1000). The following primary antibodies were used for loading controls: β-tubulin antibody (Cell Signaling Technology, 2146, 1:500); histone H2B (D2H6) rabbit mAb (Cell Signaling Technology, 12364, 1:1000). Blots were probed with goat anti-rabbit IgG H&L (HRP) preadsorbed secondary antibody (Abcam, ab97080, 1:10000). The same antibodies were used to confirm purity of cytoplasmic and nuclear fractions (Supplementary Fig. 5).