Pegfp c1 vector

For the luciferase assay, the ATG5 and ATG7 promoter regions (−1510 to −7 and −1538 to -26, respectively) were amplified from genomic DNA using the primer pairs shown in S1 Table and inserted into the pGL3.0-basic vector (Promega, Madison, USA). pOTB7-VCP and pT7T3Pac-MAP1LC3B (Korea Unigene Clone) were subcloned into the pCMV-Flag vector and pEGFP-C1 vector, respectively, and Flag-VCP was subcloned into the plenti-puro vector (Addgene) using primer pairs mentioned in S1 Table. Short hairpin RNAs (shRNAs) against KDM3B and VCP were designed using siRNA sequence design software (Clontech). Double-stranded oligonucleotides for shRNA plasmid construction were produced using primers at the 5′ and 3′ ends (S1 Table). These oligonucleotides were inserted into the AgeI/EcoRI site of the pLKO.1 TRC vector (Addgene).

The expression vectors encoding human BIS wild type (BIS-WT), human truncated BIS depleted in exon 3 (BIS-ΔE3), or exons 2–3 (BIS-ΔE2E3) were prepared by PCR amplification with primers including start and stop codons, as well as with cDNA from SRSF3-knockdown 293T cells, and subsequently inserted into a pcDNA3.1(-) vector (Addgene, Watertown, MA, USA). For the GFP-BIS wild type (WT) minigene reporter construct, the PCR products of human BIS genomic DNA, including exons 2–3 and part of the introns, were ligated into the pEGFP-C1 vector (Addgene), and exon 4 and part of the intron were ligated into pJET1.2/blunt (Thermo Fisher Scientific). Next, the first fragment with the GFP coding sequence and second fragment were ligated into the pcDNA3.1(-) vector. To generate the mutant minigene in which the SRSF3 binding site is deleted (GFP-BIS-Mut), we performed overlapping PCR reactions. A simple map for the minigene and the primers used for cloning are provided in Figure S1 and Table S2.

Mouse Kif6 (mKif6) cDNA encoding the 803 aa protein (NP_796026) was cloned from a mouse brain cDNA library and sequenced. The cDNAs of full-length (FL) mKif6 (1-803aa) and motor-less (ML) mKif6 (360-803aa) were then subcloned into pEGFP-C1 vector (Addgene plasmid #54759) or pmCherry2-C1 vector (Addgene plasmid #54563; both a gift from Michael Davidson), yielding EGFP-Kif6 FL or mCherry-Kif6 FL or mCherry-Kif6 ML, respectively. mCherry was removed and mKif6 (1-493aa)-mNeonGreen was added into vector pmCherry2-C1 yielding mKif6 (1-493)-mNG.

The coding sequence of the BAP tag (GLNDIFEAQKIEWHE) (70 (link)) was inserted after amino acid 96 of N-terminally GFP-tagged SP followed by a stop codon using site-directed mutagenesis with the CloneAmp HiFi PCR Premix (TaKaRa Bio). The BirA and ER-BirA constructs were obtained from Addgene (38 (link), 71 (link)). The N-terminal deletion mutants of GFP-SP-BAP (d10 to d60) were generated by PCR, and the products were cloned between the XhoI and BamHI sites of GFP-SP in the pEGFP-C1 vector (Addgene). Alanine scanning mutagenesis was used to prepare specific GFP-SP-BAP mutants between SP amino acids 50 to 61. In this approach, two to three amino acids in GFP-SP-BAP were replaced by alanine for each construct using the CloneAmp HiFi PCR Premix (TaKaRa Bio) according to the manufacturer.

DNA fragments encoding Suv39h1-CD (residues 39–105 or 39–96) were PCR-amplified from pCAG-IRES-Puro-FLAG-Suv39h1 (Muramatsu et al., 2016 (link)). The amplified Suv39h1-full length or -CD was cloned into the TOPO pCRII vector (Clontech) and subcloned into the bacterial expression vector pGEX-6P-3 (GE Healthcare) to make the GST-fusion molecule. The GST-Suv39h1-CD (39–96) was only used for ITC. To express FLAG-tagged wild-type or mutant Suv39h1, DNA fragments encoding FLAG-Suv39h1 were generated by PCR using pCAG-IRES-Puro-FLAG-Suv39h1 as the template, and the amplified FLAG-Suv39h1 was cloned into the TOPO pCRII vector. Suv39h1 mutagenesis was performed with the TOPO pCRII vector according to the manufacturer’s instructions (Stratagene). Next, the wild-type or mutant FLAG-Suv39h1 was subcloned into the pMCs-IRES-GFP retrovirus expression vector (Cell Biolabs). To express GFP-Suv39h1 fusion molecules, the PCR-amplified DNA of wild-type or mutant Suv39h1 was subcloned into the pEGFP-C1 vector (Addgene), and the GFP-Suv39h1 fusion part was amplified again by PCR and subcloned into the BamH1 and SalI sites of pMCs-IRES-GFP from which IRES and the vector’s own GFP sequences were deleted.

N-terminal mNeonGreen-, mRuby3-, or mEos4b-tagged mouse STING (NM_028261) was introduced into pMXs-IPuro. C-terminal HaloTag7-tagged mouse TBK1 (NM_019786), mouse GM130 (NM_001080968.2), mouse TfnR (Transferrin Receptor) (NM_011638.4), or mouse TGN38 (NM_009443.3) was introduced into pMXs-IBla. C-terminal mScarletI-tagged human TBK1 (NM_013254) was introduced into pMXs-IBla. N-terminal HaloTag7-tagged mouse Rab6a (NM_024287) or mouse Rab11a (NM_017382.5) was introduced into pMXs-IHyg. C-terminal FLAG-tagged mouse α-COP (NM_009938.4) was introduced into pMXs-IBla. The cDNA plasmid encoding N-terminal mEos4b-tagged mouse cavin1 (NM_008986.2) was produced by replacing GFP in the pEGFP-C1 vector (addgene, #68401) with mEos4b.
The iD4H (D4^{Y415A/D434S/A463W}) mutant fragment was generated by the standard two-step PCR mutagenesis technique using pCold I-GFPenvy-D4H (D4^D434S)^{66 (link)} as a template. The iD4H fragment was introduced into pMXs-IBla. To construct E. coli expression vector, mNeonGreen-iD4H fragment was amplified by PCR using pmNeonGreen-iD4H as a template and cloned into pCold I (Takara Bio, Shiga, Japan).