Guide RNA were designed to specifically cut in the first exon of CD13 gene in the human genome, Ensembl sequences ENSG00000166825. Oligos containing the guide RNA sequence were cloned into lentiCRISPR v2 (Addgene plasmid no. 52961) (63). Scrambled guide RNA was also designed as control. Guide RNAs used were as follows: 5′-CAGTGCGATGATTGTGCACA-3′ for HCD13 and 5′-CAGTCGGGCGTCATCATGAT-3′ scramble control. Lentivirus was made using the packaging plasmids psPAX2 and pMD2.G (Addgene plasmid nos. 12260 and 12259) into HEK293T (Human Embryonic Kidney 293 cells expressing large T antigen) cells using Lipofectamine 2000 (Thermo Fisher Scientific). Virus was harvested at 48 hours after transfection. Human cell lines were plated and transduced by the lentivirus. Forty-eight hours after transduction, puromycin (2 ng/μl) was added to the culture to select for lentiCRISPR integration. The region of CRISPR binding was amplified by PCR and sequenced by Sanger sequencing. Clones that were shown to have frameshifts on both alleles were expanded, confirmed, and tested further to confirm the knockout of CD13 by immunoblot and flow cytometry analysis. Plasmid pcDNA3 HA-tagged dominant-negative ARF6 T27N plasmid was purchased from Addgene. The membrane-permeant, myristoylated peptide that mimics the N terminus of ARF6, ARF6 inhibitory peptide (myr-ARF6 peptide), was purchased from MyBioSource.

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