Penter plasmid

4-morpholineethanesulfonic acid (MES), Sodium cyanoborohydride (NaBH₃CN), Tween-20, Carboxy-methoxy lamine (CMO), Proclin-300, Dimethylsulfoxide (DMSO), Biotinyl-N-hydroxy-succinimide (NHS-Biotin), protease inhibitor cocktail and PMSF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin-coated donor beads and unconjugated europium-acceptor beads were purchased from Beyondbiotech (Shanghai, China). Anti-p53 DO-1 and anti-MDM2 N-20 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-His monoclonal antibody was acquired from Bioworld Technology (St. Louis, USA). The open reading frame (ORF) encoding p53 was generated by RT-PCR amplification from 293T cells and subcloned into a pEnter plasmid (ViGene Biosciences, Rockville, USA) carrying a C-terminal 6 × his tag. GST (glutathione transferase) recombinant pGEX-4T-MDM2 plasmid was a kind gift from Mien-Chie Hung (The University of Texas, Austin, TX, USA)^{44 (link)}. The sequences of both recombinant plasmids generated were verified by sequencing prior to use. All other reagents used were of analytical reagent grade and a Milli-Q water purification system (Millipore, MA, USA) was employed to supply ultra-pure water used throughout the experiments.

The USP21 or GATA3 coding sequence was cloned into the pENTER plasmid (ViGene Biosciences Inc., Rockville, MD, United States) to overexpress USP21 or GATA3. The Flag or Myc labeled-empty plasmid vector (pENTER) was purchased from Vigene. The small interfering RNA (siRNA) targeting USP21, GATA3, or MAPK1 was designed and synthesized by RiboBio (Guangzhou, China). All transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States). For in vivo experiment, cells were seeded into a 6-well plate and cultured overnight, and an appropriate lentivirus-packing vector overexpressing USP21 synthesized by GenePharma (Shanghai, China) was added to the cells for infection.

The coding DNA sequence of HJURP was obtained from pENTER plasmid (ViGene Bio, CH826769, China) and ligated into pLenti-C-Myc-DDK-IRES-Puro (pCMV) plasmid (OriGene, USA). The shRNA targeting HJURP (shHJURP) primer was synthesized by Sangon Biotech (Shanghai, China) and cloned to pLKO.1-Puro plasmid (Addgene, USA). Plasmids were amplified in competent escherichia coli and extracted from escherichia coli using Endo-Free Plasmid Midi Kit (Omega Bio-tek, USA). Constructed plasmids were stored at -20°C for lentivirus package preparation. The sequence of shHJURP was as follows: sense 5'-CCTCGAAGTATTCTTCCTTGA-3', antisense 5'-TCAAGGAAGAATACTTCGAGGT-3'.