Ab133337

All operations were conducted according to the instructions in the kit (#EZ-Magna RIP kit 17–701). Briefly, HT22 cells were collected and incubated with 5 µg of SOX2 (ab133337; Abcam)-specific antibody or normal IgG antibody for 2 h at 4 °C after adding an equal precipitation volume of RNA immunoprecipitation (RIP) complete lysate. The cell lysate was then added to the samples and spun overnight at 4 °C to produce beads, which were then washed six times with RIPA buffer. Finally, proteinase K treatment was used to release RNA from the bound protein. RNA was isolated using phenol:chloroform:isoamyl alcohol and precipitated using ethanol for the subsequent RT-PCR analysis.

Proteins were resolved on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were blocked for one hour in PBS 5% nonfat dry milk at room temperature and primary antibodies incubated O/N at 4 °C. The primary antibodies were detected by DyLight 680 or 800 conjugated secondary antibodies (LI-COR) diluted 1:5000 in PBS Tween 0.1% and 5% nonfat dry milk and subsequently visualized with the Odyssey® Infrared Imaging System (LI-COR). The antibodies specific for MLL1 (Millipore, 05-764), Vincullin (Abcam, ab129002), H3K4me2 (Abcam, ab32356), H3K4me3 (Abcam, ab8580), H3K9me2/3 (Cell Signalling, 5327), H3 (Abcam, ab10799), MYC (Abcam, ab32072), PAX2 (Abcam, ab79389) SOX2 (Abcam, ab133337) were diluted 1:1,000 in PBS Tween 0.1% and 5% nonfat dry milk O/N at 4 °C.