β tubulin

Cells were lysed in ice-cold RIPA cell buffer (P0013B, Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail. The proteins (40–100 μg) were separated on 10–15% PAGE gels and electrotransferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk, the membrane was incubated with specific primary antibody and horseradish peroxidase-conjugated secondary antibody. The antibodies used are Flag (GNI4110-FG-S, GNI, Japan, 1:1000), HA (GNI4110-HA-S, GNI, Japan, 1:1000), Stat3 (12640S, Cell Signaling Technology, USA,1:1000), phospho-Stat3^Y705 (9131S, Cell Signaling Technology, USA, 1:1000), Socs3 (sc-73045, Santa Cruz, 1:1000), Prdm14(D221722, BBI, China, 1:1000), H3K27me3 (39055, Active Motif, China, 1:1000), H3 (4620s,Cell Signaling Technology, USA, 1:1000) and β-Tubulin (200608, ZENBIO, China, 1:2000). The band density was analyzed with ImageJ according to ImageJ User Guide. Briefly, we inverted the greyscale images and select sample bands with rectangular selections. The proteins levels were normalized with respect to the β-Tubulin level, and the grayscale ratio of protein/β-Tubulin was calculated and visualized with GraphPad Prism 8.0.