Pcmv 3tag 7

FLAG-TRAF6 (21624), FLAG-HA-USP15 (22570), and FLAG-BECN1 (24388) plasmids were purchased from Addgene. HA-tagged Ub plasmids were obtained from Dr. J. H. Shim (University of Massachusetts Medical School, USA). Using the FLAG-HA-USP15 plasmid, full-length FLAG-USP15 and MYC-USP15 constructs were cloned into a pCMV-3Tag-7 (Agilent technologies, 240202) or a pCMV-3Tag 6 vector (Agilent technologies, 240200). Using FLAG-BECN1, the full-length MYC-BECN1 construct was cloned into a pCMV-3Tag-7 vector (Agilent Technologies, 240202). Truncated mutants of MYC-BECN1 were generated as previously described [10 (link), 20 (link)–23 (link)]. MYC-USP15 C269A and MYC-USP15 H862A mutants were generated by site-directed mutagenesis as previously described [24 (link)].

To create FLAG-AQP0 and Myc-AQP0 fusion proteins, the PCR products of WT-AQP0 and Y219*-AQP0 were digested with BamHI and XhoI restriction enzymes, respectively, purified with a PCR purification kit (Tiangen Biotech, Beijing, China), and subsequently cloned into the digested mammalian expression vector, pCMV-3Tag-6(Agilent Technologies, Shanghai, China), which contained an N-terminal triple FLAG epitope tag;and pCMV-3Tag-7 (Agilent Technologies, Shanghai, China), which contained an N-terminal tripleMyc epitope tag. All of the constructs were verified by direct sequencing.

All cell lines (Bxpc-3, MIAPACA-2, PANC-1 and NIH3T3) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Except for the Bxpc-3, which was cultured in RPMI 1640 medium (Invitrogen, Carsbad, CA, USA), all other cell lines were incubated in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum at 37°C with 5% CO₂ and 95% humidity. Antibodies for ING4 and JMJD3 were bought from Abcam. Anti-TDG antibody was from Santa Cruz and anti-Fas, GAPDH, tri-methyl-H₃K₂₇, cleaved-Caspase3 and PARP were from Cell Signaling Technology (Boston, MA, USA). All chemical inhibitors were bought from Sigma. K-Ras (1-688bp) full length open reading frame (ORF) was amplified by PCR using the GoTaq Green Master Mix (M7123, Promega, Madison, WI, USA) with cDNA reversing transcription from total RNA of PNAC-1 cells. The primers used were listed in Table 1. The PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). After sequence verification, the inserts were sub-cloned using BamHI and XhoI restriction sites into a mammalian expression vector pCMV-3Tag-7 (Agilent, La Jolla, CA, USA).
TDG (1-1233bp) and ING4 (1-747bp) full length ORF inserted into neomycin resistant mammalian expression vector EX-Z4461-M14 was purchased from GeneCopoeia (Rockville, MD, USA). H-Ras (Q61L) cDNA was purchased from Upstate (Lake Placid, NY, USA).