Pegfp c1

Mouse neuroblastoma Neuro2a (N2a) and human glioblastoma A172 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics and incubated at 37 °C in the presence of 5% CO₂. Stable cells expressing shRNAs were established by transfection of shHuD plasmid (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or control plasmid under puromycin (Invitrogen™, Waltham, MA, USA) selection. Enhanced green fluorescent protein (EGFP) reporter was prepared by cloning the 3′UTR sequence of Ccl2 mRNA (533–806, 274 nt) into the pEGFP-C1 (BD Bioscience, Franklin Lakes, NJ, USA) vector. HuD overexpression plasmids (pHuD) were received as a gift from Prof. Alessandro Quattrone [18 (link)]. Transfection of small interfering RNAs (HuD siRNA (siHuD) and control siRNA (siCtrl)) (Genolution Pharmaceuticals, Inc., Seoul, South Korea) or plasmids were achieved using Lipofectamine™ 2000 (Invitrogen™) according to the manufacturer’s instructions.

The human RTTN cDNA (accession number, BC156291) was purchased from the IMAGE clone consortium (IMAGE clone No. 100061722) and subcloned into pEGFP-N2 (BD Biosciences Clontech) or pFlag-CMV2 (Sigma-Aldrich). To generate the GST-fusion constructs, the cDNAs encoding various potion of RTTN were fused in-frame to the GST-encoding sequence in the pGEX4T vector (GE Healthcare). The RTTN mutant constructs were generated by site-directed mutagenesis using a QuikChange kit (Stratagene). The expression constructs encoding various GFP-tagged RTTN mutant proteins were generated using pcDNA4/To/myc-His-A (Invitrogen) or pLVX-Tight-Puro (BD Biosciences Clontech) vectors. Sequencing was used to confirm all generated plasmids. The cDNAs encoding full-length CP110, H2B and STIL were obtained by RT-PCR using total RNAs from human HEK293T cells and subcloned in-flame into a pEGFP-C1 (BD Biosciences Clontech) or a pLVX-Tight-Puro (BD Biosciences Clontech) vector. The cDNA constructs for GFP-tagged full-length STIL, SAS-6, and CPAP and various Flag-tagged STIL truncated mutants were from ref. ^{8 (link)}. The cDNA constructs containing various CEP295 fragments were from ref. ^{19 (link)}.

cDNA of human eEF1A1 gene were amplified from total mRNA of human fetal liver (BiocolorBioScience& Technology, Shanghai, China) by reverse transcription-polymerase chain reaction (RT-PCR). The primers used in PCR were designed as 5′-CTGAATTCATGGGAAAGGAAAAGACT-3′ and 5′-ACTGGATCCTTTAGCCTTCTGAGCTTTCTG-3′. The eEF1A1 cDNA were cloned into pEGFP-C1 (BD Clontech, Palo Alto, CA) and verified by PCR, restriction enzyme digestion and DNA sequencing (Invitrogen).

Human Bcl-xLRFP plasmid was a gift from Richard J. Youle (National Institutes of Health, Bethesda, MD). NFLGFP was a gift from Freddy Radtke (École Polytechnique Federale de Lausanne (EPFL), Switzerland). pBABE, pBABE-NIC-NLS and pBABE-NIC-NES were kind gifts from B.A. Osborne (University of Massachusetts/Amherst, MA, USA). D1ER and 2mtD3cpv probe were a kind gift from Roger Y Tsien (42 (link), 54 (link)).
NIC1-GFP was prepared by sub-cloning NIC1 gifted by J. Aster (Harvard Medical School, Boston) into pEGFP-C1 (BD Clontech, CA, USA). NIC1-RFP has been described earlier (5 (link)). sJagged was obtained from Upstate Biotechnology (MA, USA). Human Grp75 Tagged ORF Clone was obtained from Origene (RG201397, MD, USA) and sub-cloned into pBABE vector. Following primers were used for sub-cloning:
Construct sequences were verified by automated Sanger sequencing conducted in-house.