CRISPR guide RNA (gRNA) was obtained for VPS35, Cav1, and Cltc from Sigma-Aldrich and transfected using protocols from our previous publications (9, 93). After transfection, cells were incubated for 48 hours before treatment. The lentivirus-based CRISPR-Cas9 KO plasmid against gene Vps35, with target site 5′-CAAGTCATTTCCTCAATCCAGG-3′ in a U6gRNA-Cas9-2A-RFP vector, was purchased from Sigma-Aldrich. The lentivirus-based CRISPR-Cas9 plasmids, pLV-U6g-EPCG-Cav1 and pLV-U6g-EPCG-Cltc with the Cav1 and Cltc gRNA target sequences 5′-GTTGAGATGCTTGGGGTCGCGG-3′ and 5′-TACTGAAGCCAATGTTTGCTGG-3′, respectively, were purchased from Sigma-Aldrich. To make the lentivirus, the lenti–CRISPR-Cas9 Vps35 KO plasmid and nontarget control plasmid were transfected into 293FT cells using the MISSION Lentiviral Packaging Mix from Sigma-Aldrich according to the manufacturer’s instructions. The lentivirus was harvested 48 hours after transfection and added to the microglial cell line at a multiplicity of infection of 100 to KD VPS35, caveolin-1, and clathrin expression.

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