Ab97030

LV tissue samples were ground in liquid nitrogen and sonicated in PBS with 1X phosphatase and protease inhibitors. The protein content in the sample was quantified using the BCA protein assay kit (ThermoFisher Scientific, 23225). Then, 40µg protein was loaded on each well and separated using SDS-PAGE and transferred into methanol-activated PVDF membrane. The membrane was then blocked with 5% non-fat milk in TBST for 1 hour. Then, the membrane was incubated with primary antibodies for cardiac β-myosin heavy chain (Abcam, ab50967 at 1:1000 dilution), atrial natriuretic peptide (Thermo Fisher Scientific, PA5-29559 at 1:1000 dilution), and GAPDH (Cell Signaling Technologies Inc., CST 97166 at 1:1000 dilution) overnight at 4°C. Next day, the membrane was washed with TBST three times, 10 minutes each. The membrane was then incubated with HRP-conjugated secondary antibodies (Abcam ab97064, ab97030 at 1:4000 dilution in 5% non-fat milk in TBST) for 1 hour at room temperature. Then, the membrane was washed with TBST three times, 10 minutes each to remove unbound secondary antibodies. The protein band was detected using an iBright FL1500 instrument (Thermo Fisher Scientific) and the bands were quantified using ImageJ analysis software from the National Institutes of Health.

Western blot was performed as previously described57 (link). In brief, 50-μg of total protein from the L4 stage worms was fractionated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with the mouse-derived anti-GAPDH antibody (60004-1-Ig, Proteintech) and donkey anti-mouse IgG antibody (ab97030, Abcam). Actin was probed with anti-ACTIN antibody (60008-1-Ig, Proteintech) as an internal control. Blots were treated with Western Bright ECL (WBF25, Gel Company, USA) and examined by the chemiluminescence System (Image Station 4000 mm, Kodak, USA). All of the experiments were repeated at least three times.