Western blots

The protocol of the SDS–PAGE and western blotting was as follows:

SDS–PAGE and western blotting

Various mouse tissues including whole hearts were first homogenized in lysis buffer (20 mM Tris, 1% NP-40, 2 mM EGTA, detergents (including ionic detergents: sodium dodecyl sulfate and 100 mmol/L sodium deoxycholate, and nonionic detergent Nonidet P-40), and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA) pH 7.4) for 30 min at ice. Lysates of the mouse cardiac tissues were centrifuged for 15 min at 14,000 g at 4˚C. Protein concentration of the mouse cardiac tissues was quantified by employing the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Equivalent protein quantities (40-60 μg) of the mouse tissues were subjected to analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to nitrocellulose (NC) membranes. Membranes mentioned above were blocked with fat-free milk in TBS-T buffer (5%) for 120 min and incubated with the primary antibodies of the phosphorylated-FUNDC1 (1:1000) and total-FUNDC1 (1:1000) polyclonal antibodies overnight at 4˚C, which were produced by immunizing rabbits with synthesized and purified phosphorylated and nonphosphorylated peptides from FUNDC1 (Abgent, SuZhou, China) according to our previous study (Nature Cell Biology. 2012; 14:177–185). After probing with the indicated primary antibodies, the NC membranes were incubated with the appropriate HRP-conjugated secondary antibodies (KPL) for 180 min at 4˚C. Representative blots of the tissues including cardiac tissues were illustrated from three times of independent experiments and the images were taken with an enhanced chemiluminescence (ECL) reagent system as described previously (Nature Cell Biology. 2012; 14:177–185). For the internal control of cardiac tissues, alpha-actin (not beta-actin) was employed. GAPDH or tubulin also could be used for loading control.

Polyclonal antibodies against FUNDC1 were generated by immunizing rabbits with recombinant FUNDC1 (δ transmembrane domain) protein produced in Escherichia coli using pET28a expression vector (Anti-FUNDC1 polyclonal antibody (1:1,000; AVIVA).). Anti-p-FUNDC1 (Tyr 18) polyclonal antibody (1:1,000) was generated by immunizing rabbits with purified phosphopeptides in FUNDC1 and affinity purified (Abgent, SuZhou, China), as described previously (Nature Cell Biology. 2012; 14:177–185). All these two antibodies were employed in the eLIFE paper according to a previous study in our group (Nature Cell Biology. 2012; 14:177–185).

For the samples of cardiac tissues and cardiomyocytes, the commercial antibodies against FUNDC1 and p-FUNDC1 were also available now: anti-FUNDC1 (#NBP1-81063, Novus Biologicals, LLC) and anti-FUNDC1 (#ABC506, EMD Millipore Corporation). Weilin Zhang from the Institute of Zoology, Chinese Academy of Sciences, Beijing, China, states that he has no conflict of interests with the companies mentioned above. He personally points out the information on the antibodies to provide help to the research field, but he was not supported by them.

In our eLIFE paper, the information on the antibodies was included in the section of “Reagents, antibodies” as the followings: Anti-p-FUNDC1 (Tyr 18) (1:500) and anti-FUNDC1 (1:1,1000) polyclonal antibodies were produced by immunizing rabbits with synthesized and purified phosphorylated and non-phosphorylated peptides from FUNDC1 (Abgent, SuZhou, China).

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