Pqe32 vector

Point mutations were introduced by using the QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany). The pQE32 vector (Qiagen, Hilden, Germany) with the mRuby cDNA cloned into the BamHI-XbaI sites was used as a template. Primers were ordered from Biomers (Ulm, Germany).
To generate mGarnet fusion constructs, the codon-optimized cDNA for mammalian expression of mGarnet was PCR amplified using primers containing the appropriate restriction enzyme sites. For N-terminal fusions, the mGarnet PCR products and the pcDNA3.1 vectors containing the gene sequence of the fusion partners were digested with XhoI and XbaI (human histone 2B (H2B)), PvuI and NotI (human α-actinin), EcoRI and XbaI (human cytochrome c oxidase subunit VIII, i.e., the mitochondrial targeting sequence mito) and XhoI and XbaI (LifeAct (F-actin marker)). RBP-J Interacting and tubulin-associated (RITA) protein and α-tubulin were fused to the C-terminal end of mGarnet via KpnI and EcoRI and NheI and XbaI, respectively. Ligation was performed using the Quick Ligation Kit (NEB, Frankfurt am Main, Germany). Ligation products were transformed and amplified in E. coli XL1 and purified using the Pure Yield^TM Plasmid Miniprep System Kit (Promega, Mannheim, Germany). DNA Sequencing was carried out by GATC Biotech AG (Konstanz, Germany).

TNF-Fc, HF-TNFSF10/TRAIL and FASLG-Fc were expressed and purified as described before [15 (link),57 (link),58 (link)]. In short, the cDNAs encoding the extracellular portion of human TNF (AA78–233) or human FASLG/CD95L (AA117–281) were fused at the N terminus to the constant region (Fc) of human IgG1 (CH2-CH3, AA102–329), preceded by the TNFRSF10B signal peptide (AA1–55). The expression vector pcDNA3.1 (Invitrogen, V79020) was transfected in HEK293T cells and the supernatants containing the Fc-tagged proteins were used for the experiments shown. HF-TNFSF10/TRAIL was produced by cloning the extracellular portion of human TNFSF10/TRAIL (AA95–281) in the pQE32 vector (Qiagen, N32915) preceded by a FLAG and 6×His tag. The protein was expressed in M15 bacteria (XL Biotech, CHC00032) and purified using Ni-NTA (Cytiva, 29048631).

Frozen Symbiodinium sp. KB8 cells were ground to a fine powder under liquid nitrogen using a mortar and pestle. Total RNA in the frozen powder was purified with RNeasy Mini kit reagents (Qiagen, Tokyo, Japan). cDNA was synthesized with PrimeScript 1st strand cDNA Synthesis kit reagents (Takara, Shiga, Japan) and total RNA as the template. To obtain the first half of the VLKP gene, the forward primer was 5'-ATGAACGCGGCCACGGCCTTTG-3' and the reverse primer was 5'-TCAAATAACGATGGCTGTCTCTTCAGCC-3'. RT-PCR was carried out using KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan) and the PrimeScript-synthesized cDNA as the template. Using In-Fusion HD Cloning kit reagents (Takara), the reverse-transcribed amplicons were inserted into a SmaI-digested pQE-32 vector downstream of an encoded, in-frame, N-terminal His₆ tag (Qiagen). Sequencing of the plasmid confirmed correct insertion of the amplicon.