Phospho smad1 5ser463 465

Western blot analyses were performed as previously described (Jiang et al., 2016 (link); Jiang et al., 2017a ; Jiang et al., 2017b (link)). The HDAC Antibody Sampler Kit (#9928), Phospho-HDAC4^Ser246/HDAC5^Ser259/HDAC7^Ser155 (#3443), HDAC2 (#5113), HDAC6 (#7558), H3K4me2 (#9725), H3K4me3 (#9727), H3K27me2 (#9728), H3K27me3 (#9733), Phospho-AKT^Ser473 (#4060), AKT (#4685), Phospho-mTOR^Ser2448 (#5536), mTOR (#2983), Phospho-AMPKα^Thr172 (#2535), AMPKα (#5831), Phospho-MEK1/2^Ser217/221 (#9154), Phospho-ERK1/2^{Thr202/Tyr204} (#4370), ERK1/2 (#4695), Phospho-JNK1/2 ^{Thr183/Tyr185} (#4668), JNK1/2 (#9258), Phospho-p38^{Thr180/Tyr182} (#4511), p38 (#8690), Phospho-β-Catenin^Ser675 (#4176), Non-phospho-β-Catenin^{Ser33/37/Thr41} (#8814), β-Catenin (#8480), Phospho-IKKα/β^Ser176/180 (#2697), IKKβ (#8943), Phospho-NF-κB p65^Ser536 (#3033), NF-κB p65 (#8242), Phospho-Smad2^Ser465/467 (#3108), Smad2 (#5339), Phospho-Smad3^Ser423/425 (#9520), Smad3 (#9523), Phospho-Smad1/5^Ser463/465 (#9516), and Smad1 (#6944) antibodies were obtained from Cell Signaling Technology. Antibodies against Histone H3 (ab1791), H3K36me2 (ab9049), H3K36me3 (ab9050), H4K20me3 (ab9053), H3K9me1 (ab9045), H3K9me2 (ab1220), H3K4me1 (ab8895), H3K23me1 (ab176132), H4K5ac (ab51997), H4K8ac (ab15823), H3K18ac (ab40888), H3K23ac (ab61234) and H4K12ac (ab46983) were purchased from Abcam.

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.