Fusion sl2

Differentiated L6 myoblasts treated with OFS were lysed, and equal amounts of protein were subjected to SDS-PAGE and immunoblotted with antibodies specific for AMPK, p-AMPK (Thr 172), ACC, p-ACC, JNK, p-JNK, ERK, p-ERK, p38, p-p38, GLUT4 and β-actin (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive bands were visualized using chemiluminescent imaging system (Fusion SL2, Vilber Lourmat, Marne-la-Vallée Cedex, France), and analyzed by Bio1d software (Vilber Lourmat, Marne-la-Vallée Cedex, France).

The procedures for western blotting were described in our previous reports [21 (link), 23 (link)]. The following primary antibodies were used for western blotting: ERα (1:500 dilution, Abcam), ERK (1:1000 dilution, Cell Signaling Technology), p-ERK (1:1000 dilution, Cell Signaling Technology), and β-actin (1:5000 dilution, Abcam). The secondary antibody was obtained from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). Briefly, the cells were homogenized at 13,000 rpm for 5 min at 4 °C, and the protein was obtained. The protein (20 μg) was subjected to SDS-PAGE, and electro-transferred to PVDF membrane (Whatman, Maidstone, Kent, UK). The membranes were incubated with different primary antibodies and then secondary antibodies then filmed using a chemiluminescent imaging system (Fusion SL2, Vilber Lourmat, Marne-la-Vallée Cedex, France). The optical density of the protein band was quantified by the ImageJ software (Bethesda, USA). The density of ERα was shown as the relative value to that of β-actin, while the density of p-ERK was shown as the relative value to that of its total kinase, ERK.

An antibody against SLR1 and an anti-ACTIN polyclonal antibody were purchased from Cosmo Bio (Cat. No. CT-NU-001-1; Tokyo, Japan) and Agrisera (Cat. No. As13 2640; Vännäs, Sweden), respectively. OsPP2C08-OX and wild-type (WT) seedlings were grown on half-strength MS medium containing GA₃ or ABA. Whole shoots were ground in liquid N₂, and total protein was extracted using an extraction buffer containing 0.05 M Tris–HCl (pH 7.4), 0.2% (w/v) SDS, 5% (v/v) glycerol, 1.5% (v/v) Triton X-100, 1% (v/v) β-mercaptoethanol, 1 mM EDTA, 1 mM dithiothreitol, and 1X complete Mini EDTA-free Protease Inhibitor (Roche, Rotkreuz, Switzerland). Immunoblotting was performed as described previously [24 (link)]. Peroxidase activity was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). Proteins were detected with Fusion SL2 (Vilber Lourmat, Eberhardzell, Germany).