Hikari signal enhancer solution

A total of 1 × 10⁶ cells were plated in 6-well plates, and different concentrations of MBE, MN, and HK were added. Total cell lysates (30 µg) were extracted using mPER buffer (Cat# 78501; Thermo Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Cat# 4693116001; Roche, Basel, Switzerland). All antibodies were diluted using HIKARI signal enhancer solution (Cat# 02267-41, NACALAI, Kyoto, Japan). Molecular weight markers (Bio-Rad, Hercules, CA, USA) were loaded onto 4–20% TGX gels (Cat# 4561094, Bio-Rad) and run for 100 min at 100 V. The proteins were then transferred onto a nitrocellulose membrane (Cat#2001, Bio-Rad, CA, USA). The antibodies used are listed in Supplementary Table S1. All approximate molecular weights were decided by comparison with the migration of pre-stained protein standards. A ChemiDoc MP imaging system was used to produce digital images (Bio-Rad, Hercules, CA, USA). The quantitative protein band was performed using ImageJ software (National Institutes of Health, Rockville, MD, USA) and was shown in Supplementary Figures.

A total of 1 × 10⁶ cells was plated in 6-well plates, and, then, different concentrations of PNF were added. Total cell lysates (50 µg) were extracted using mPER buffer (Cat# 78501, Thermo Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Cat# 4693116001, Roche, Basel, Swiss). Isolated TA muscles were extracted using tPER buffer (Cat# 75810, Thermo Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail. All antibodies were diluted using the HIKARI signal enhancer solution (Cat# 02267-41, NACALAI, Kyoto, Japan). The antibodies used are listed in Table S2. A Chemidoc MP imaging system was used to produce digital images (Bio-Rad, Hercules, CA, USA). The Image J version 1.52a software was used to quantify the relative protein intensity. The densitometric values of target proteins were normalized to endogenous tubulin and GAPDH levels.