Nonfat skim milk

All cell lines were harvested at 80-90% confluence and then lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, #89901). Protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, #23225). Protein samples were resolved by 8-10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, #162-0117). The membrane was blocked in Tris-buffered saline (TBS) containing 0.05% Tween-20 (Amresco, 0777-1L) with 5% nonfat skim milk (BD, #232100) for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4°C. After three washes in TBS with Tween-20 (TBST), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. After three washes in TBST, the membrane was treated with the EZ-ECL kit (Biological Industries, #20-500-120) and visualized using the Tanon-5200 multi-automatic chemiluminescence/fluorescence imaging analysis system (Tanon Science & Technology Inc). The following antibodies were used: anti-HEY1 (Abcam, ab154077), anti-β-actin (CST, #3700), and secondary antibodies including anti-rabbit IgG, HRP-linked antibody (CST, #7074) and anti-mouse IgG, HRP-linked antibody (CST, #7076).

Following the treatment, the myotubes were homogenized in RIPA buffer supplemented with phosphatase and protease inhibitors (Millipore, Burlington, MA, USA) for 30 min. Protein lysates were then separated using an 8% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel) and then transferred onto a polyvinylidene difluoride membrane (Millipore) at 100 V for 90 min. The membrane was blocked in 5% nonfat skim milk (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at RT. Subsequently, the membrane was incubated with primary antibodies (Table 1) and then with secondary antibodies for 2 h at RT. Protein bands were visualized on an Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden) imaging system using an enhanced chemiluminescence (ECL) system (Bio-Rad, Hercules, CA, USA). Quantification of protein levels was performed using ImageJ (NIH, Bethesda, Maryland, USA).
Table 1

Primary antibodies used for western blot and immunocytochemistry

Antibody	Company	Product no.	Dilution
Myosin Heavy Chain (MHC)	R &D Systems (Minneapolis, Minnesota, USA)	MAB4470	1:500
Atrogin-1	Abcam (Cambridge, Cambridgeshire, U.K)	Ab168372	1:1000
MURF1	Invitrogen (Waltham, Massachusetts, USA)	PA5-76695	1:1000