Phosphatase inhibitors cocktail

Cells were lysed in NP-40 buffer (P0013F, Beyotime, Shanghai) supplemented with phosphatase inhibitors cocktail (Bimake, America) and protease inhibitors cocktail (Bimake, America). The protein concentrations were measured using Nanodrop (Thermo Fisher) with Bio-Rad protein assay reagent. For immunoprecipitation analysis, the lysates (1000–3000 μg) were incubated with 50% slurry of Protein A/G sepharose beads (GE Healthcare) conjugated antibody for 3–5 h at 4 °C. After washed four times with NETN buffer (0.5% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl and 1 mM EDTA), the recovered immuno-complexes were resolved by SDS–PAGE and immunoblotted with indicated antibodies.

Tissues or cardiomyocytes were sonicated in RIPA lysis buffer containing 1% NonidetP-40, 0.1% SDS, protease inhibitor, while adding phosphatase inhibitors cocktail (Bimake, Houston, USA), and then homogenized. The nuclear and cytosolic protein were extracted by commercially available kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells expanded sufficiently under low osmotic pressure, then the cell membrane ruptured, releasing cytoplasmic proteins. Nucleus precipitation could be centrifuged to obtain after then. Finally, the nuclear protein was extracted by high salt nucleoprotein extraction reagent. Equal quantities of tissues or cardiomyocytes protein lysates were subjected to SDS-PAGE (Bio-Rad), and transferred onto PVDF membrane. Blocking was made at room temperature with 5% nonfat milk powder prepared in Tris-buffered saline containing 0.1% Tween 20. Then, membranes were incubated overnight at 4 °C with corresponding antibodies, and followed by incubation with appropriate secondary HRP-conjugated antibodies. Antibodies against p38 (#8690), P-p38 (#4511), NFκB-p65 (#8242), ERK (#4695), P-ERK (#4370), P-Stat3 (#9145), Stat3 (#4904) and GAPDH (#2118) were obtained from Cell signaling Technology (Danvers, USA). Antibodies against FNDC5 (ab174833), P-JAK2 (ab195055), JAK2 (ab108596), Nox2 (ab129068), Nox4 (ab154244) were obtained from Abcam (Cambridge, UK).

Cells were lysed using NP-40 buffer (P0013F, Beyotime, Shanghai) supplemented with phosphatase inhibitors cocktail (Bimake, USA) and protease inhibitors cocktail (Bimake, USA). The lysates were incubated with 20 μL HA/Flag beads (Bimake, USA) overnight at 4 °C. After being washed 4 times with NETN buffer (NP-40, EDTA, Tris.8.0 and NaCl), HA/Flag beads were mixed with loading buffer and heated denaturing the proteins at 100 °C. Finally, Western blotting assay was next performed.