Sin3b

Total protein was extracted from cells in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulphate) supplemented with a protease and phosphatase inhibitor cocktail; protein lysate was combined with SDS loading buffer (4% SDS, 0.2% bromophenol blue, 20% glycerol, 5% 2-mercaptoethanol) and boiled for 5 minutes. Protein samples were run on Bis-Tris (Novex) gels and transferred to nitrocellulose. Blots were blocked in 50% Seablock blocking buffer (Thermo Scientific, diluted in PBS) and incubated with primary antibody overnight in blocking buffer. Antibodies used were to HCC1/RBM39 (Atlas, HPA001591), SIN3B (Santa Cruz, sc-768), Phospho-SMAD1/5 (Ser463/465) (Cell Signaling Technologies, 9516) and beta tubulin (Sigma T5283). Secondary antibodies used were IRDye 680RD Donkey anti-Rabbit IgG and IRDye 800CW Donkey anti-Mouse IgG (LI-COR), incubated for 1 hour in blocking buffer. Blots were imaged on a LI-COR Odyssey CLx. Nuclear and cytoplasmic fractions were extracted with an NE-PER kit (Thermo Scientific), to the manufacturer’s instructions. Separation of cellular fractions was confirmed by blotting for anti-Histone H3 (Abcam, 131711). Phosphorylated and unphosphorylated fractions from C2C12 cells were extracted with a PhosphoProtein Purification Kit (Qiagen) to the manufacturer’s instructions.