Pcmv ha vector

The p2.1 reporter was purchased from ATCC and is commonly used for monitoring HIF-1α transcriptional activity [34 (link)]. Hypoxia response element (HRE) reporter and SOD2 promoter luciferase reporter were provided by Navdeep Chandel and Xin-Hua Feng, respectively. The HRE-reporter contains three hypoxia response element (HRE) repeats and is commonly used for monitoring HIF-1α transcriptional activity [35 (link)]. The wild-type EPEC NleB gene was originally obtained from Feng Shao, and its enzymatically dead mutant (D221/223A) was PCR amplified and subcloned into the pCS2-EGFP vector and pGEX-4T vector. Wild-type human HIF-1α was subcloned into the pCMV-Myc vector and pCMV-HA vector (Clontech). Human HIF-1α-330-399aa was cloned into the pET32a vector. All R/K mutants of HIF-1α were subcloned into the pCMV-Myc vector. Wild-type human HIF-2α was subcloned into the pCMV-Myc vector and the pCMV-HA vector. Human HIF-1α domains were subcloned into the pCMV-Myc vector.

Expression constructs in the pCMV-HA vector or pCMV-myc vectors (Clontech, Mountain View, CA, USA) encoding mouse and human HA-IFITM3, human myc-IFITM3, and mouse HA-IRGM1 have been previously described [4 (link),20 (link),21 (link)]. Untagged human IFITM3 was generated by removal of the HA tag from the pCMV-HA vector by site directed mutagenesis. mEmerald-Cathepsin B was a kind gift from Michael Davidson via Addgene (plasmid #54024). Human codon-optimized DNA sequences were generated from the amino acid sequences for MAV IFITM (UniProt A0QLS9) and MAB IFITM (UniProt B1MKV5). These sequences were purchased as synthetic Gene Strings from Life Technologies (Carlsbad, CA, USA). The complete sequences that were ordered including EcoRI and SalI restriction sites are shown below. These sequences were inserted into the pCMV-HA vector in frame with the HA tag via restriction sites. Phylogenetic tree analysis of the mycobacterial IFITMs, alignments, and calculations of percent amino acid identities were performed using Clustal Omega software (University College Dublin, Dublin, Ireland).

For the preparation of purified STING and TRIM29, HEK293T cells (ATCC CRL-3216) were transfected with an expression plasmids encoding full-length or truncated versions of HA- or Myc-tagged STING or TRIM29. All the above plasmids were constructed in pCMV-Myc or pCMV-HA vector (Catalog: 631604, Clontech). Lysates were prepared from the transfected cells, followed by incubation with anti-HA or anti-Myc beads. Proteins were eluted from the beads after beads were washed six times with PBS. For precipitation with anti-HA or anti-Myc beads, purified HA-tagged WT STING or truncations of STING were incubated for 2 h with purified Myc-tagged TRIM29 or purified HA-tagged TRIM29 or truncations of TRIM29 were incubated for 2 h with purified Myc-tagged STING. Beads were added; after 2 h of incubation, the bound complexes were pelleted by centrifugation. Proteins and beads were analyzed by immunoblot analysis with anti-HA or anti-Myc Abs. Uncropped scans of immunoblots are provided as Supplementary Fig. 15.

To map the interacting domains between 3D^pol and Prp8, the full-length and various truncated forms of human Prp8 were amplified by PCR from the human Prp8-pCMV-XL5 cDNA clone (OriGene) using specific primers. The PCR product was inserted into a pCMV-HA vector (Clontech) between the XhoI and NotI sites to enable the expression of HA-tagged proteins. The EV71 full-length infection cDNA clone was used to amplify full-length and various truncated forms of EV71 3D^pol by PCR, followed by cloning into the EcoRI and KpnI sites of the p3XFLAG-Myc-CMV-25 vector (Sigma) to enable expression of the EV71 3D^pol constructs as fusions with 3 adjacent FLAG epitopes. To overexpress these proteins, 2 µg of the constructs of the various truncated forms of Prp8 and 3D^pol was co-transfected into HEK293T cells (1×10⁶/per 6-well plate) using Lipofectamine 2000 reagent (Invitrogen) for 48 h. The cells were harvested for FLAG-IP using a FLAG-immunoprecipitation kit (Sigma). After lysis and centrifugation, the supernatant was treated with 10 µg/ml RNase A at 30°C for 1 h, and then 40 µl of anti-FLAG M2 agarose affinity gel was added at 4°C for 12 h. Proteins were then eluted by competition with 3×FLAG peptide. In the WB assay, the precipitated proteins were identified using an anti-HA antibody (diluted 1∶5000; Sigma) and an anti-FLAG antibody (diluted 1∶5000; Sigma).

Plasmids pHA-XBP1s, pHA-ATF6, pHA-eIF2α, pHA-G3BP1, and pHA-TIA1 were constructed using conventional cloning techniques. The RT-PCR primers and the corresponding restriction sites (underlined) are as follows: XBP1s-F (5′-GGAAGATCTATGGTGGTGGTGGCAGCG-3′ [BglII]), XBP1s-R (5′-AAAAGCGGCCGCTTAGACACTAATCAGCTGGGGG-3′ [NotI]), eIF2α-wt-F (5′-CCGGAATTCATGCCGGGGCTAAGTTGTAGA-3′ [EcoRI]), eIF2α-wt-R (5′-AAAAGCGGCCGCTTAATCTTCAGCTTTGGCTTCC-3′ [NotI]), ATF6-F (5′-CCGCTCGAGATGGAGTCGCCTTTTAGTCC-3′ [XhoI]), ATF6-R (5′-AAAAGCGGCCGCCTACTGCAACGACTCAGGGAT-3′ [NotI]), G3BP1-F (5′-CGCGGATCCATGGTTATGGAGAAGCCT-3′ [BamHI]), G3BP1-R (5′-ACGCGTCGACTCACTGCCTTGGAGTTGT-3′ [SalI]), TIA1-F (5′-CGCGGATCCATGGAGGACGAGATGCCCAA-3′ [BamHI]), and TIA1-R (5′-ACGCGTCGACTCACTGGGTTTCATACCCGG-3′ [SalI]). The target fragments were inserted into a pCMV-HA vector (Clontech) using the corresponding restriction enzyme cutting sites mentioned above. pHA-eIF2α-S51A that introduces the Ser51Ala substitution for eIF2α was generated using the Fast mutagenesis system (catalog number FM111-01; Transgen, Beijing, China) (P1, 5′-TCTTCTTAGTGAATTAGCCAGACGACGTAT-3′; P2, 5′-CTAATTCACTAAGAAGAATCATGCCTTCAA-3′). The recombinant plasmids were transfected into N2a cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA).