Characterizing LDLR Regulation by EXOC4 and EXOC8

HuH7 cells were cultured in DMEM containing 10% FBS and penicillin/streptomycin. Cellular uptake assays were performed with fluorescent conjugates of LDL (Cayman Chemical, Ann Arbor MI, 10011229) or transferrin (ThermoFisher Scientific, Waltham MA, T35352). For immunoblotting, membranes were probed with antibodies against LDLR (Abcam, Cambridge UK, ab52818, 1:2000), β-actin (Santa Cruz Biotechnology, Dallas TX, sc-47778, 1:5000), EXOC4 (Abcam, ab205945, 1:1000), and EXOC8 (Santa Cruz, sc-515532, 1:500). For flow cytometry, cells were stained with a fluorescently-conjugated antibody against LDLR (R&D Systems, Minneapolois MN, FAB2148G). CRISPR-mediated gene disruption was performed by cloning gRNA sequences into BsmBI sites of pLentiCRISPRv2 (Addgene #52961, a gift from Feng Zhang[17 (link)]) or into BbsI sites of pX459 (Addgene #62988, a gift from Feng Zhang). Genotyping was performed by Sanger sequencing of PCR amplicons of genomic target sites with individual alleles deconvoluted by TIDE analysis of chromatograms[33 (link)]. Lentiviral expression constructs were generated by assembly of cDNA sequences (GE Healthcare Dharmacon, Lafayette CO, EXOC4 MHS6278-202759993 and EXOC8 MHS6278-202758964) and a blasticidin resistance cassette into the LeGO-ic2 plasmid (Addgene # 27345, a gift from Boris Fehse[34 (link)]) using HiFi DNA Assembly Master Mix (NEB, Ipswich MA).