P3xflag cmv 7

pDONR223-TNK1 was a gift from William Hahn & David Root (Addgene plasmid # 23894). For HEK 293T expression of N-terminal FLAG-tagged Tnk1 (UniProtKB - Q13470-2), the Tnk1 coding sequence was amplified by PCR and subcloned into p3xFLAG-CMV-7.1 (Sigma) using EcoRI and BamHI restriction sites. Tnk1 mutants were generated by site-directed mutagenesis using the Agilent QuikChange II kit. For expression in HeLa cells, Tnk1 DNAs were subcloned into pEGFP-C1 (BD Biosciences Clontech) using EcoRI and BamHI restriction sites. For baculoviral expression of Tnk1^ΔCT and Tnk1^ΔSAMΔCT in Sf9 cells, we first subcloned the DNA sequence encoding a GST-tag and PreScission cleavage site sequence from the pGEX-6p-1 (Millipore Sigma) into the baculoviral expression vector pFastBacHTa (ThermoFisher) using EcoRI and NotI restriction sites. We used PCR to amplify the sequences encoding Tnk1^ΔCT (residues 1–510) and Tnk1^ΔSAMΔCT (residues 111–510) and ligated them into the modified pFastBacHTa vector backbone using XbaI and KpnI restriction sites.

The pLKO.1 DNA plasmids containing the shRNA sequence against human TPMS (TCHP) was purchased from Sigma Aldrich (Mission^®RNAi TRCN0000127662 and TRCN TRCN0000130868). The scrambled sequence shRNA plasmid was purchased from Addgene, plasmid #1864. The packaging plasmids used were pCMV-ΔR8.2Δvpr, plasmid #8455 and pCMV-VSVG, plasmid #8454 from Addgene. Retroviral particles were generated by transient transfection of the vector-histone H2B into phoenix-ampho cells by using calcium phosphate method. The resulting supernatant was used to infect Hela and HCT116 cells. To generate an expression plasmid for 3xFLAG–tagged, the full-length TpMs coding sequence was amplified by PCR with primers having the sequence 5′-GATGACAAGCTTGGAAACTCCGAGCCTCAGAGA-3′ and 5′ GGATCCTCTAGATTCTCTGTACTTATGGTACCC-3′. The PCR product was digested with HindIII and XbaI, and the resulting DNA fragment was inserted into p3xFLAG-CMV-7.1 (Sigma Aldrich) to prepare p3xFLAG-TCHP. A 3xFLAG–TPMS (TCHP) coding sequence was then amplified by PCR, the products were purified by gel and verified by sequencing. siRNA for TpMs are SMARTpool: ON-TARGETplus TCHP siRNA (Dharmacon).

The Huh7.5 cell line (a generous gift from Dr. Charles M. Rice, The Rockefeller University, New York, NY, USA) and the Huh7/FT3-7 cell line (a generous gift from Dr. Stanley M. Lemon, UNC-Chapel Hill, Chapel Hill, NC, USA) are human hepatocellular carcinoma cell lines that are highly permissive for replication of sub-genomic and full-length HCV RNA [36 (link)].
pCDNA3.1-Hygro-NS3/4A and pCDNA3.1-Hygro-Core for the expression of NS3/4A or Core, respectively, were generous gifts from Prof. Itai Benhar, Tel-Aviv University, Tel-Aviv, Israel. p3X-Flag-CMV-7.1 (Sigma) and the cDNA for human T-cell protein tyrosine phosphatase (TC-PTP) were kindly provided by Dr. Georg Bode, Department of Gastroenterology, Hepatology, and Infectiology, Heinrich-Heine-University, Düsseldorf, Germany. The cDNA of human TC-PTP was amplified and subcloned into the p3X-Flag-CMV-7.1 expression vector.

Antibodies and sources: CDK9 (1.1000), pCDK9 (Thr187) (1.1000), RNAPII (1.1000), phospho-RNAPII(Ser2) (1.1000), PARP (1.1000), and NUP98 (1.1000) (Cell Signaling Technology); HEXIM1 (1.1000), NELF-a (1.1000), LARP-7 (1.1000), c-Myc (1.1000), SPT5 (1.1000), and GAPDH (1.5000) (Santa Cruz Biotechnology, Dallas, TX, USA); BRD4 (1.1000) (Abcam, Waltham, MA, USA); Caspase-8 (1.1000) (Enzo Life Sciences, Farmingdale, NY, USA); β-Actin (1.10000), (Sigma-Aldrich, St. Louis, MO, USA); Cyclin T (1.1000) (Bethyl, Montgomery, TX, USA). Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assays (Promega, Madison, WI, USA); AnnexinV and 7AAD (BD); BAY1251152, BI894999, ABBV744, Paclitaxel and Carboplatin (Selleckchem, Houston, TX, USA); BioCoat Matrigel invasion chamber (Corning, Corning, NY, USA); Migration chamber (Ibidi, Gräfelfing, Germany); RNeasy Plus kit (Qiagen, Venlo, The Netherlands). The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene, Watertown, MA, USA); p3xFlag-CMV-7.1 (E7533, Sigma, Ronkonkoma, NY, USA).

The full-length open reading frame of the human PLD3 gene or the human GRN gene, selected as a candidate for PLD3 interactors, was amplified by PCR using PfuTurbo DNA polymerase (Agilent Technologies, Palo Alto, CA, USA) and the set of sense and antisense primers. Subsequently, PCR products were cloned in the expression vector named p3XFLAG-CMV7.1 (Sigma), pCMV-Myc (Clontech, Mountain View, CA, USA), pDsRed-Express-C1 (Clontech), or pZsGreen1-N1 (Clontech) to express a fusion protein with an N-terminal Flag tag, Myc tag, DsRed tag, or C-terminal ZsGreen tag, respectively. The vectors were transfected in HEK293 cells, HeLa cells or SK-N-SH cells using Lipofectamine 2000 reagent (Invitrogen) for transient expression. The M232 isoform of PLD3 was prepared from the cloned vector using QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). After cotransfection of the vectors, the protein extract was processed for immunoprecipitation with mouse monoclonal anti-Flag M2 affinity gel (Sigma) or rabbit polyclonal anti-Myc-conjugated agarose (Sigma), followed by western blot with a rabbit polyclonal anti-Myc antibody (Sigma) or a mouse monoclonal anti-FLAG M2 antibody (Sigma), respectively.

Murine wild-type p21 cDNA (Origene) was cloned into p3XFLAG-CMV 7.1 (Sigma). Additionally, p21 was tagged with a 3× HA tag by PCR mutagenesis. p21^K7R was generated by PCR mutagenesis. p21^ΔPCNA, p21^Δdegron and p21^K7RΔPIP (Q139A, L142A, F145A, Y146A) mutants were generated by site-directed mutagenesis (Agilent). FLAG and HA-tagged wild-type and mutant p21 were cloned into pDONR221 (Invitrogen) and pINDUCER20 using BP and LR clonase kits (Invitrogen) respectively. pINDUCER reagents were a gift from Guang Hu at NIH/NIEHS. DNA transfection was performed using LipoD293 (Signagen) or Lipofectamine (Invitrogen).