Western blot analysis

Mouse and rat LV tissue was harvested and lysed. Protein concentrations were determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., 23227). Proteins were separated by electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked in Tris-buffered saline containing Tween-20 (pH 7.6) and 5% nonfat dry milk for 2 h, and subsequently incubated overnight at 4 °C with primary antibodies to the following proteins: Bcl-2 (#3498), Bax (#5023), GRP78 (#3177), caspase-12 (#2202), PKA (#4782), p-PKA (Thr197), AMPK (#2532), p-AMPK (Thr172, #2531), CREB (#9197), p-CREB (Ser133, #9198), Akt (#2967), p-Akt (Thr308, #13038), ERK1/2 (#4696), p-ERK1/2 (Thr202/Tyr204, #9106), Na⁺-K⁺-ATPase α1 (#23565), β-actin (#8457) (all from Cell Signaling Technology, Inc.), CTRP9 (LifeSpan Biosciences, Inc., LS-C373857), CRT (Abcam, ab22683 and Santa Cruz, sc-373863), AdiopR1 (Abcam, ab126611), Calnexin (Santa Cruz, sc-23954), KDEL ER marker (Santa Cruz, sc-58774), and GAPDH (CMCTAG, Inc., AT0002). After washing, the membranes were probed with appropriate secondary antibodies (Zhongshan Company, ZB-2301, ZB-2305) at room temperature for 90 min. Protein bands were detected by Bio-Rad Imaging System (Hercules), and normalized to β-actin or GAPDH.