Pmscv

HCMV US7 and US8 constructs were fused at the N-terminus to a hemagglutinin (HA) tag containing N-terminal H2-K^b signal sequences. We then generated the C-terminal deletion constructs of HA-US7 (HA-US7ΔCT) and HA-US8 (HA-US8ΔCT) by PCR and the point mutant HA-US8 (HA-US8 Mut; Y204A/Y211A) by site-directed mutagenesis. All of the N-terminal HA-tagged US7 and US8 or C-terminal HA-US12-US16 genes into pcDNA3.1 or pMSCV, respectively (Invitrogen, San Diego, CA). In addition, HA-US7 and HA-US8 were fused at the C-terminus to green fluorescence protein (GFP) by subcloning them into a pEGFP N3 vector (Clontech, Palo Alto, CA). We also sub-cloned all of the above constructs, including TLR4-Myc, TLR3-Myc, MD2-Myc, Flag-TLR4, UNC93B1-GFP, into a retroviral vector pMSCV (Clontech) or a pLHCX vector (Clontech). We verified all constructs by sequencing. All primer sequences are listed in Supplementary Table 1.

The miR-500a-3p expression plasmid was generated by cloning the genomic pre–miR-500a-3p gene, with a 300-bp sequence on each flanking side, into retroviral transfer plasmid pMSCV-puro (Clontech Laboratories Inc.) to generate plasmid pMSCV- miR-500a-3p. pMSCV- miR-500a-3p was cotransfected with the pIK packaging plasmid in 293FT cells using the standard calcium phosphate transfection method, as previously described [25 (link)]. Thirty-six hours after the co-transfection, supernatants (MOI: 20) were collected and incubated with cells to be infected for 24 h in the presence of polybrene (2.5 μg/ml). After infection, puromycin (1.5 μg/ml) was used to select stably transduced cells over a 10-day period. The 3’UTR of SOCS2, SOCS4 and PTPN11 were amplified and cloned downstream to the luciferase gene in a modified pGL3 control vector (Promega, USA), and the list of primers used in clone reactions was presented in Additional file 1: Table S1. Anti-miR-500a-3p, small interfering RNA (siRNA) for SOCS2, SOCS4 and PTPN11 knockdown and respective control RNA (50 nM) were synthesized and purified by RiboBio. The sequence of anti-miR-500a-3p is cagaauccuugcccaggugcau. Transfection of miRNAs, siRNAs, and plasmids was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions.

Constructs encoding GFP-, GST- and Flag-tagged mouse RBPJ were generated by PCR amplification and cloned into pLenti-PGK [46] (link) or pMSCV (Clontech) for expression in mammalian cells, and in pFastBac1 for expression insect cells. The cDNA encoding the mouse RBPJ isoform 2 (NP_001074396.1) was obtained from Thermo Scientific. For protein expression in SF9 cells, Flag-tagged RBPJ was purified using M2-affinity chromatography [47] (link). RBPJ mutations were generated with the QuikChange Site-Directed Mutagenesis Kit (Agilent).
For generating anti-mRBPJ antibodies, GST-mRBPJ was expressed in SF9 cells and purified using glutathione sepharose chromatography. The rabbit anti-RBPJ polyclonal antibody was generated by Cocalico Biologicals, Inc. Characterization of the anti-RBPJ antibody is shown in Figure S4.

p85α cDNA was cloned upstream of an internal entry site (IRES) and the enhanced green fluorescence protein (EGFP) in the bicistronic retroviral plasmid, pMIEG3 [37 (link)]. Alternatively, p85α was tagged on the C-terminal end with yellow fluorescent protein (YFP) and cloned into the retroviral plasmid, pMSCV (Clontech). Ecotropic retrovirus containing the vectors was used to transduce Pik3r1^−/− fetal liver cells, cells were sorted for EGFP or YFP positivity, and differentiated into neutrophils. Data using both constructs are combined for statistical analyses, as the YFP tag did not alter the function of p85α (data not shown).

Individual gRNAs were cloned into lentiCRISPR v2 (Addgene 52961) or lentiGuide-Puro (Addgene 52963) and cDNAs were cloned in pLenti (Addgene #22255), pMSCV (Clontech), pAAV TBG (Adddgene #105535), or pcDNA4TO (Addgene #60914). Drug resistance genes in lentiGuide, pLenti, and pMSCV were replaced by Blasticidin S, Neomycin or Hygromycin-resistant genes. pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) were used for lentiviral packaging. pMD2.G and gag/pol (Addgene #14887) were used for retroviral packaging. All gRNA and gene information is provided in Supplementary Data 2.
LDTS of perilipin 1 was obtained from mouse Plin1 cDNA (Dharmacon, Clone Id:4166741) and LDTS of mouse perilipin 5, 4BNC and human PNPLA5 were synthesized (gBlocks, Integrated DNA technologies). eLIR and LIR mut fragments were obtained by PCR using primers containing mutations. PCR template was a plasmid containing human OPTN (Addgene #27052). PCR fragments were inserted into backbone plasmids using In-Fusion HD Cloning Kit (Takara) or NEBuilder HiFi DNA Assembly Master Mix (NEB). For AAV generation, mCherry, LDTS, and eLIR or LIR mut PCR fragments were inserted in the NotI-BamHI site of pAAV TBG plasmid (Adddgene #105535). The resulting plasmids were verified by sequencing.