Ab3110

Antibodies recognizing β-Actin (Abclonal, AC026, RRID: AB_2768234), H3 (abcam, ab1791, RRID: AB_302613), H3K27ac (abcam, ab4729, RRID: AB_2118291), H3K4me1 (Cell Signaling Technology, #5326, RRID: AB_10695148), p53 (Santa Cruz, sc-126, RRID: AB_628082), p21 (Cell Signaling Technology, #2947, RRID: AB_823586), MDM2 (abcam, ab3110, RRID: AB_303518), PUMA (Cell Signaling Technology, #4976, RRID: AB_2064551), MLL3(Merck ABE1851) and TNS3 (Abclonal A7991, RRID: AB_2772674) were purchased from indicated vendors. PCR primers were custom synthesized by TSINGKE and siRNAs by GenePharma. Nutlin-3A (S8059) was purchased from Selleck.
MDA-MB-231 and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) at 37 °C with 5% CO₂, and U2OS and 769-P cells in RPMI 1640 (Gibco). Both mediums were supplemented with 10% FBS (Biological Industries) and 1% penicillin-streptomycin solution (HyClone). All cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences.

Proteins were extracted from mouse tissues and cells using RIPA buffer containing fresh protease and phosphatase inhibitors, and quantified using a protein assay (Bio-Rad Laboratories, Hercules, CA). Protein samples (30 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was conducted using antibodies against PTPRO (Proteintech group, #12161), LC3 (Cell signaling technology, #12741), p62 (Cell signaling technology, #5114), PI3K (Cell signaling technology, #4249), p-PI3K(Y458/Y199) (Bioworld, BS4605), p-AKT(S473) (Cell signaling technology, #9271), p-AKT(T308) (Cell signaling technology, #9275), AKT (Cell signaling technology, #9272), p-s6k (Cell signaling technology, #9205), s6k (Cell signaling technology, #9202), p-MDM4(S367) (Abcam, ab122926), MDM4 (Abcam, ab76362), MDM2 (Abcam, ab3110), p53 (Cell signaling technology, #2527), Cyclin D1 (Santa Cruz, sc-20044), Bcl-2 (Santa Cruz, sc-130307). The results were visualized using a chemiluminescent detection system (Pierce ECL substrate western blot detection system, Thermo Scientific, 32106) and exposure to autoradiography film (Kodak XAR film).