Western blotting was performed using a previously described protocol [31 (link)]. The seeded cells were removed from the culture dishes and centrifuged, and the resulting cell pellets were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). The lysates were mixed with sample buffer (Sigma, St. Louis, MO, USA) at a 1:1 ratio and then resolved on SDS–PAGE gels. After electrophoresis, the proteins were transferred to a PVDF membrane; the membrane was incubated with the primary antibody against GDF15 (1:1000, rabbit monoclonal antibody, no. 8479; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight, anti-rabbit IgG secondary antibody (1:1000, no. 7074; Cell Signaling Technology) for one hour at room temperature, and an anti-β-actin polyclonal antibody (MBL, Tokyo, Japan) for one hour at room temperature. Blots were visualized using an Amersham Imager 600 (Cytiva, Tokyo, Japan) and Immobilon Clasico (Sigma–Aldrich, St. Louis, MO, USA). The immunoblots were evaluated by quantifying the band intensity using ImageJ software.
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