Hysk1

pcDNA3.1-v5-hYSK1 and deletion fragments of hYSK1 were generated by PCR using the human cDNA clone-hYSK1 (Origene Technologies, MD, USA) as a template. The PCR product was purified, digested with EcoRI/XhoI, and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen, MA, USA). For TOPO-GFP-hYSK1, the PCR product was inserted into the pcDNA3.1-NT-GFP-TOPO vector (Invitrogen, MA, USA). The GST-p16^INK4a and GST-deletion fragments of p16^INK4a were inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, PA, USA). For pDsRed-p16^INK4a, the PCR product was cloned into the ApeI site of the pDsRed vector (Clontech Laboratories, CA, USA). HA-SP-1 was cloned into the EcoRI/XhoI site of the pCMV-HA and pCMV-Myc vectors (Clontech Laboratories, CA, USA). The shRNA-hYSK1 plasmid was constructed into the pSilencer 4.1-CMV-hyg vector (Ambion, NY, USA). The pGL2-p16-luc vector was a gift from Dr. Gordon Peters (Cancer Research UK, London) and the pGL2-MMP-2-luc vector was a gift from Dr. Etty N. Benveniste (The University of Alabama at Birmingham, Birmingham, AL). Various expression vectors were amplified in E. coli XL1-blue or BL21 cells and plasmids were purified using a Qiagen midi kit (Qiagen, Hilden, Germany). The DNA sequences of all plasmids were confirmed by sequencing (Dye Terminator ABI Type Seq., Bionex, NJ, USA)