β tubulin

Western blotting assay was used to detect the protein expression of Nestin, β-tubulin, Wnt1, β-Catenin and phosphorylase of β-Catenin (Phospho-Tyr489). After treated with AMD3100, the cellular lysates of RASNF-01001 were obtained using RIPA lysis buffer containing 6 μg/ml PMSF. Total protein was extracted and the protein concentration was determined by Bradford assay. Western blotting assay was carried out using antibodies against Nestin (Signalway Antibody, USA), β-tubulin (Signalway Antibody, USA), Wnt1 (Abcam, USA), β-catenin and phosphorylase β-catenin (Phospho-Tyr489) (Signalway Antibody, USA). The immune complexes were measured with pro-light HRP Kit (TIAN GEN, China). Expression of proteins was analyzed using ImageJ software (NIH, Bethesda, MD, USA). All the experiments were repeated three times over multiple days.

The antibody against DPY30 used for IHC was from Abcam (ab126352). The primary antibodies used for WB and IHC included: PCNA (Santa Cruz, sc-25280), Ki67 (Santa Cruz, sc-23900), cyclin A (Santa Cruz, sc-271682). And other antibodies applied to WB as follows: α-actinin (Proteintech, 11313-2-AP), β-tubulin (Signalway Antibody, #48885), H3K4me3 (CST, C42D8, #9751), Histone H3 (CST, D1H2, #4499), cyclin B1(sc-245), cyclin D1 (sc-8396), cyclin E (sc-377100), p21 (sc-6246). Anti-mouse or rabbit secondary antibodies were purchased from Sigma. FBS, high glucose DMEM medium, and Trypsin (with EDTA) were purchased from Gibco (Thermo Fisher Scientific, USA). penicillin-streptomycin purchased from BasalMedia. Other reagents were all analytical grade. Other relevant kits or special supplies will be described below.