U2af65

Binding reactions were the same as in vitro splicing, except for omission of polyvinylalcohol. Reactions were irradiated with 254-nm UV light (Ultralum) on ice for 10 min and treated with a mixture of RNase A (0.05 U/1 μl) and RNase T1 (2U/μl; Ambion) at 30°C for 20 min. An equal volume of 2x Laemmli buffer was added and samples were boiled for 5 min before loading on to 10% SDS-PAGEs. The gels were fixed, dried and exposed to PhosphorImager screens. For immunoprecipitation, 50 μl of cross-linked reactions were mixed with 400 μl of the IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.05% NP-40), 50 μl of protein G-coupled Sepharose beads and the indicated antibodies. Antibodies were purchased from Sigma (U2AF65, hnRNP A2/B1), ABcam (Tra2β, SRSF1) or were a generous gift from Professors Douglas Black (hnRNP H), UCLA, and Gideon Dreyfuss (hnRNP A1), University of Pennsylvania. After incubation at 4°C for 2 hrs, the samples were washed 5 times with the IP buffer. Beads were resuspended in 20 ul of the Laemmli buffer, boiled for 5 min and loaded on a 10% SDS-PAGE.

We performed immunohistochemistry assay using UltraSensitive TM SP IHC Kit (MaxVision Biotechnology, Fuzhou, China) according to manufacturer’s protocol. In brief, paraffin-embedded sections were processed using the IHC Kit and subsequently incubated with primary antibodies against Ki-67 (1:1000; ProteinTech), TGF-B1(1:100; Abcam), RREB1(1:100; Abcam), U2AF65(1:100; Abcam) at 4 °C overnight. The sections were incubated with a secondary antibody from the IHC Kit for 30 min at 37 ˚C and then imaged under a light microscope (Olympus).

RIPA lysis with PMSF and 8 M Urea were used to extract protein of GSC cell lines. The nuclear and cytoplasmic proteins were extracted separately using a NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). The protein concentration was detected by BCA method and 15-30ug protein was loaded per well in and separated by SDS-PAGE. The polyvinylidene difluoride (PVDF) membranes with 0.22um pore size were indicated primary antibodies against U2AF65 (1:1000; Abcam), RREB1 (1:1000; Abcam), NESTIN (1:1000; Abcam), OCT4(1:1000; Abcam),SOX2 (1:1000; Abcam), Nanog (1:1000; Abcam), CD133 (1:1000; Abcam), TGF-B1 (1:1000;Cell Signaling Technology), SMAD2 (1:1000; Cell Signaling Technology), pSMAD2 (1:1000; Cell Signaling Technology), SMAD3(1:1000; Cell Signaling Technology), pSMAD3(1:1000; Cell Signaling Technology), lamin B1(1:1000; Proteintech), β-actin(1:1000; Proteintech). Following 1 h incubation with secondary antibody (ProteinTech), the signals were examined by chemiluminescence Femto-sig ECL kit (Tanon, Shanghai, China).