Et1602 18

Total protein was extracted from frozen heart tissue using a Minute™ muscle tissue total protein extraction kit (SA-06-MS, Invent Biotechnologies, Inc., United States) following the manufacturer’s instructions. Protein samples were quantified using a BCA Protein Assay Kit (E-BC-K318-M, Elabscience, Wuhan, China). Protein samples were resolved on 10% SDS-PAGE gels (PG212, EpiZyme, Shanghai, China) and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, United States). The membranes were blocked with 5% fat-free milk and incubated overnight at 4°C with primary antibodies (ANP, 1:1000, ab209232, Abcam, United States; β-MHC, 1:1000, 22280-1-AP, Proteintech, China; p38, 1:1000, ET1702-65, Huabio, China; JNK, 1:1000, RT1550, Huabio, China; ERK1/2, 1:1000, 67170-1-Ig, Proteintech, China; p-p38, 1:1000, ER1903-01, Huabio, China; p-JNK, 1:1000, 4668S, Cell Signaling Technology, USA; p-ERK1/2, 1:1000, 4370T, Cell Signaling Technology, United States; DUSP 6, 1:1000, ET1602-18, Huabio, China; β-ACTIN, 1:1000, ab8226, Abcam, United States). Then, the membranes were washed and incubated with secondary antibodies (Goat anti-Mouse IgG, 1:10000, ab216776, Abcam; Goat Anti-Rabbit IgG, 1:10000, ab6721, Abcam). The protein bands were visualized by ECL (Millipore) and quantified using ImageJ software (National Institutes of Health).

Paraffin-embedded heart tissue was sectioned into 5μm sections. The paraffin sections were deparaffinized and rehydrated in xylol and alcohol. The antigen was retrieved in a microwave oven with citrate antigen retrieval solution (ZLI 9064, ZSGB-BIO, Beijing, China) after three washes with phosphate-buffered saline (PBS, G0002-2L, Servicebio). Sections were incubated overnight at 4°C with primary antibodies (Collagen I, 1:500, ab270993, Abcam, Cambridge, MA, United States; Collagen III, 1:200, ab7778, Abcam; DUSP6, 1:100, ET1602-18, Huabio, China; p-ERK1/2, 1:200, 4,370T, Cell Signaling Technology, Danvers, MA, United States), followed by three washes with PBS. Sections were then incubated with a universal two-step detection kit (PV-9000, ZSGB-BIO) following the manufacturer’s instructions. After three washes with PBS, the sections were stained with diamino-benzidine (DAB, ZLI-9018, ZSGB-BIO) and hematoxylin and then dehydrated, and transparency were performed. Finally, the sections were made into digital slides with Pannoramic Digital Slide Scanners (Pannoramic DESK, P-MIDI, P250, and P1000, 3DHISTECH Ltd.).