Pegfp n2

CERKLa-GFP was obtained by cloning the coding region of hCERKL532 cDNA (NM_201548.4) in pEGFPN2 (BD Bioscience, NJ, USA), by using XhoI and BamHI restriction sites. Mitochondrial-targeted DsRed (mitoDsRed) was kindly provided by Prof. Eduardo Soriano (University of Barcelona, Barcelona, Spain).

All human cell lines and their in vitro requirements were obtained from ATCC and tested in an exponential growth phase (Manassas, VA, USA) (Table S1A,B). Construction of pCMV-p14, pCMV-tBID, pCMV-p14-tBID, and pCMV-p14-GFP vectors cDNA for the human p14 and tBID were generated by RT-PCR with primer sets derived from sequences found in the NCI Nucleotide Database. The GFP gene was generated by PCR using pEGFP-N2 (BD Biosciences Clontech, Palo Alto, CA, USA) as a template. The fragments were inserted into the corresponding multiple cloning sites A or B, or both cloning sites of the vector pIRES (BD Biosciences Clontech, Palo Alto, CA, USA). This was utilized to make corresponding vectors pCMV-p14, pCMV-tBID, pCMV-p14-tBID, and pCMV-p14-GFP, as shown in Figure 1A.

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)}.