Opti 4cntm substrate kit

Western blot of CCDC186 protein was performed in muscle biopsy of P1.1 and in fibroblasts of P1.2, as well as control tissues. Material from P2.1 was not available. Muscle extracts were prepared in tissue extraction buffer (250 mM mannitol, 75 mM sucrose, 10 nM Tris-HCl, 0.1 mM EDTA) and centrifuged at 600 g at 4 °C for 20 min. Fibroblasts were lysed using RIPA buffer containing a protease inhibitor cocktail (1862209, Merck, Darmstadt, Germany). Briefly, cells were scraped in RIPA lysis buffer, incubated on ice for 10 min, and centrifuged at 10,000 g at 4 °C for 10 min. Protein extracts (40 ug) were subjected to SDS-PAGE, electroblotted, and visualized by immunostaining with specific antibodies followed by colorimetric detection (Opti-4CNTM Substrate Kit, Bio-Rad, Hercules, CA, USA). The antibodies used in this study were: anti-CCDC186 (HPA018019, Sigma Aldrich, Burlington, MA, USA) and anti-beta actin (ab115777, Abcam, UK). Membrane images were quantified using ImageJ software 1.53t.

Cells from patient No. 2 were treated with or without indicated inhibitors, washed with cold PBS and lysed in cell lysis buffer with a cocktail of protease inhibitors (Sigma) for 30 min on ice. Aliquots of 40 µg total proteins of lysed cells were separated with SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 3% BSA and 0.5% goat serum, and incubated with Rabbit anti-human PDGFRA monoclonal antibody (clone D1E1E, Cell Signaling) or Rabbit anti-human p44/p42 MAPK monoclonal antibody (clone 137F5, Cell Signaling) or Rabbit anti-human Phospho-p44/p42 MAPK monoclonal antibody (clone 20G11, Cell Signaling) at 4°C overnight. Subsequently, the membrane was washed with PBS and incubated with goat anti-mouse IgG-HPR and visualized with Opti-4CNTM substrate kit (BIO-RAD).