Protein Expression Analysis in DM1 Cells

Total cell proteins were prepared from the DM1 model cells, as previously described (Nakamori et al., 2017 (link)). Then, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the following primary antibodies: mouse anti-IGFBP3 (1:500; MAB305, R&D Systems), rabbit anti-PAI-1 (1:2000; NBP1-19773, Novus), rabbit anti-AKT (1:500; GTX121937, GeneTex), rabbit anti-phospho-Akt (Ser473) (1:500; 4,058, Cell Signaling Technology), rabbit anti-p53 (1:100; 2,527, Cell Signaling Technology), rabbit anti-p21 (1:1,000; 2,947, Cell Signaling Technology), rabbit anti-p16 (1:1,000; ab108349, Abcam), and rabbit anti-GAPDH (1:1,000; G9545, Sigma-Aldrich). After incubation, the immunoblots were washed, incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG (GE Healthcare), and detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare) using a ChemiDoc Touch Imaging System (Bio-Rad).