Quickblock western blocking buffer

The PTC cells were subjected to protein extraction through RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (Merck, Germany). After separating the proteins using SDS‒PAGE, they were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked for 60 min with QuickBlock™ Western Blocking Buffer (P0252, Beyotime Biotechnology, Shanghai, China) and subjected to incubation overnight at 4 °C. After five washes with TBST for 5 min, the secondary antibody combined with horseradish peroxidase (HRP) was applied for incubation at RT for one hour, and the protein was detected with BeyoECL Moon reagent (Beyotime Biotechnology, Shanghai, China).

Cells were lysed using mammalian protein extraction reagent (Beyotime, China) with protease inhibitor (ROCHE, Switzerland) and phosphatase inhibitor (ROCHE, Switzerland). Subsequently, protein concentration was determined by bicinchoninic acid protein assay kit (Beyotime, China). Next, 5 × SDS loading buffer (Beyotime, China) was added into the protein solutions and heated for 15 min at 95 °C. Then, 15 mg of protein was used for electrophoresis (60 V, 30 min; 110 V, 70 min) and transfer (200 mA, 120 min). Subsequently, blocking using QuickBlock™ Western blocking buffer (Beyotime, China) and antibody incubation were performed. The primary and secondary antibodies used are shown in the Supplementary Table 1.

Total protein from cultured myotubes was extracted using RIPA lysis buffer (Beyotime) with PMSF protease inhibitor (Beyotime), after incubation for 15 min on ice and centrifugation at 13,000× g for 10 min at 4 °C, the supernatant was collected. The proteins were separated in SDS-PAGE gels and transferred into polyvinylidene fluoride membranes (Bio-Rad Laboratories Inc.), subsequently blocked in QuickBlock™ Western blocking buffer (Beyotime). The membranes were then incubated overnight at 4 °C with primary antibodies: anti-MyH1A (F59, DHSB, 1:100), anti-MyH7B (S58, DHSB, 1:100), anti-Nfix (2D3, DHSB, 1:50), anti-β-actin (AF5003, Beyotime, 1:1000). Then membranes were washed in PBS and incubated with appropriate secondary antibodies conjugated with Dylight 680 (BS10037, BS10038, Bioworld, Nanjing, China, 1:1000) for 1 h at room temperature. The blots were developed using the Odyssey Fc imaging system (LI-COR, NE, USA).

Cells were lysed using cell lysis buffer (ThermoFisher Scientific, China) containing the protease inhibitor PMSF to prevent protein degradation. After measuring the protein concentration, 5× protein buffer was added and the proteins were denatured in a 98°C water bath. The protein samples were separated on 12% SDS–PAGE Gel SuperQuick Preparation Kit (Beyotime, China), alongside the prestained color protein marker (ThermoFisher Scientific, China). The proteins on the gel were transferred to PVDF membranes (Millipore, China) using a Trans-blot Turbo (Bio-Rad, China), and then blocked with QuickBlock™ Western Blocking Buffer (Beyotime, China). Membranes were cut, based on the bands of the protein marker, immediately after blocking. The excised membranes were incubated overnight at 4°C with PEDV N antibody (Medgene Labs, Brookings, SD, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ABclonal, China). The corresponding HRP-conjugated goat anti-mouse and anti-rabbit IgG (H + L) secondary antibodies were added the next day, followed by incubation for 1 h at room temperature. After elution, imaging was performed on a Bio-Rad imager with diluted NcmECL Ultra (NCM, China).

Extracted proteins were quantified using a BCA Protein Assay kit (Cat# 23,227, Thermo Fisher Scientific), and the samples were loaded onto SDS-PAGE using 4–20% gradient gels (Cat# M00656, GenScript Biotech), followed by transfer to a PVDF membrane (Cat# ISEQ00010, Merck Millipore). After the transfer, the membrane was blocked with QuickBlock™ Western Blocking Buffer (Cat# P0252, Beyotime Biotech) for 1 h at room temperature and then used for immunoblotting. Antibodies used in study included an anti-occludin antibody (Cat# ab167161, Abcam), an anti-ZO-1 antibody (Cat# 21773-1-AP, Proteintech), and an anti-β-actin antibody (Cat# 66009-1-1 g, Proteintech), an anti-TSP1 antibody (Cat# sc-59,887, Santa Cruz Biotechnology), and an anti-transferrin antibody (Cat# ab278498, Abcam).