Pmx ires gfp

The coding sequence for mouse Lamtor1, dominant-negative mouse LXRα (ref. 24 (link)) (1–435 amino acid residues), and dominant-negative mouse LXRβ (ref. 24 (link)) (1–437 amino acid residues) were cloned into pMX-IRES-GFP (Cell Biolabs, CA, USA). The coding sequence for murine nuclear form SREBP1 or SREBP2 (1–480 and 1–473 amino acid residues, respectively) was fused with the sequence for FLAG-tag and cloned into pRetroX-TetOne (Clontech, CA, USA). All plasmid vectors were checked with Sanger sequencing to exclude mutations. To prepare retroviral vectors, Platinum-E cells (Cell Biolabs) were used as packaging cells. BMDMs were cultured as described above, and culture supernatant of Platinum-E cells containing retroviral vector was added on day 3. For selection of BMDMs harbouring the doxycycline-inducible SREBP1 or SREBP2 genes, empty pMX-IRES-GFP retroviral vector was simultaneously infected as a fluorescent marker. On day 6, gene-transferred macrophages were sorted as GFP^high cells, which were clearly distinct from the GFP−negative population, using a FACS Aria III (BD Biosciences, CA, USA). If necessary, 200 ng ml⁻¹ of doxycycline was added to the culture media during M2 polarization after sorting. See Supplementary Fig. 8e for the confirmation of the protein expression from pRetroX-TetOne vector.

Retroviral vector pMXs human AID-, AIDΔE5- and AID7.3-ires-GFP, as well as mouse AID-GFP, have been described^{24 (link),26 (link),36 (link)}. Human AID fusions were assembled as BamHI-AID-EcoRI-Linker-HindIII-AID-E58A-XhoI cassettes into pTrcHisA (ThermoFisher) or pMX-ires-GFP (Cell Biolabs). The linker encoded a flexible (SGGGG)x3 peptide. Human SPT5 was PCR amplified and cloned into a gateway-compatible mCherry-LacRep destination vector^{56 (link)} (a gift from Dr. D. Durocher, University of Toronto, Canada). Mouse AID and Linker-BirA* were PCR amplified using gateway-compatible primers and cloned into appropriate donor vectors to generate AID-BirA* fusions into a homemade gateway-compatible pMX-ires-GFP bearing a gateway cassette cloned BamHI-EcoRI by using Multisite gateway technology (Invitrogen). AID variants were generated by PCR amplification with ad hoc oligonucleotides or by quick-change site-directed mutagenesis using KOD1 DNA polymerase (Toyobo Inc.). For oligonucleotide sequences, see Supplementary Table 2.