Nbp2 30348h

Protein pellets were obtained from skin fibroblasts derived from the IP and controls (C1, C2). Preparations of total cell proteins, including cell lysis, protein extractions, and protein quantifications were made as previously described [25 (link)]. After that, protein samples (30 µg each) were separated with a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (pore size: 0.45 µm, Merck Millipore, Burlington, Massachusetts, USA) by applying 45 V for 110 min using a wet blotting system (Mini Trans-Blot Cell, Bio Rad, Hercules, California, USA). Membranes were blocked for 1 h with tris-buffered saline with 0.1% Tween 20 (TBST) and 5% BSA. The overnight incubation with primary antibodies (Anti-RBM10/S1, Abcam ab72423, 1:300 and Anti-GAPDH, Merck Millipore, MAB374, 1:100) was performed at 4 °C. The PVDF-membrane was incubated with horse radish peroxidase (HRP)-linked secondary antibodies (Novus Biological, NBP2-30348H and NB7539, both 1:5000) at room temperature for 1 h and with enhanced chemiluminescence solution (ECL, Thermo Fisher Scientific). All antibodies were initially diluted in TBST with 5% BSA. The WB-signals of RBM10 proteins were visualized with ChemiDoc MP Imaging System (Bio Rad).