To generate Δp62 HeLa cells, a guide DNA sequence targeting exon 1 of the human SQSTM1 gene (5′-GTCATCCTTCACGTAGGACA-3′) was cloned into the Bsm BI site of the LentiCrisprV2 plasmid, which was a gift from F. Zhang (Addgene plasmid #52961), to generate pLentiV2-p62-gRNA (guide RNA). HeLa cells were transiently transfected with the plasmid and selected with puromycin for 1 day. Clonal cells were isolated, and loss of p62 was verified by Western blot and phenotypic analysis. HeLa cells that underwent similar selection but were negative and therefore expressed WT p62 were used as negative controls. Sequencing of p62KO cells confirmed deletion and frameshift mutations that introduced stop codons within the gRNA region. Δp62 cells were stably transduced with retroviral plasmids expressing Flag-tagged p62 variants or YFP as indicated.

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