Duolink proximity ligation in situ assay

Proximity Ligation Assay (PLA) was carried out using the Duolink In Situ kit (Sigma-Aldrich, Cat# DUO92101), following manufacturer’s instructions. Briefly, WT primary myoblasts were seeded on laminin-511-coated chamber-slides (m-Slide well, Ibidi, Cat# 80,826) and incubated at 37 °C and 5% CO₂. After 48 h, cells were fixed in 4% paraformaldehyde for 10 min at RT, washed in PBS and permeabilized with 0.05% Triton X-100 in PBS for 10 min at RT. Unspecific binding sites were blocked by incubating cells in 5% BSA in PBS for 30′ at RT. Cells were then incubated overnight at 4 °C with primary antibodies for COUP-TFII (Abcam, Cat# ab41859) and THRA (Sigma Aldrich, Cat# SAB4502968), in diluent buffer. Detection (Ligation and Amplification) was performed according to the manufacturer's protocol. Images were acquired with a Zeiss LSM 710 Confocal Microscope (Zeiss, Germany). Protein–protein interactions appear as red dots.