Characterization of Knockout Cell Lines

We used the following previously published cell lines: HEK293T cells (American Type Culture Collection, cat. no. CRL-3216), hTERT immortalized RPE‐1 (RPE1) wild-type cells (American Type Culture Collection, cat. no. CRL-4000), RPE1 AKAP450 knockout and RPE1 AKAP450/CDK5RAP2/MMG triple knockout cell lines^{47 (link)}, and RPE1 wild-type and RPE1 AKAP450 knockout transgenic cell lines stably expressing GFP–CDK5RAP2 (ref. ^{72 (link)}). All these cell lines were cultured in Dulbecco’s modified Eagle medium/Ham’s F10 media (1:1) supplemented with 10% fetal calf serum and 1% antibiotics (penicillin and streptomycin). The cell lines used were not found in the commonly misidentified cell lines database maintained by the Immunization Coalition of Los Angeles County. No further cell line authentication was performed. The cell lines were routinely checked for mycoplasma contamination using the LT07–518 Mycoalert assay. Polyethylenimine ‘Max’ (PEI Max, Polysciences) was used to transfect HEK293T cells with plasmids for Strep-Tactin- and streptavidin-based protein purification at a 3:1 ratio of PEI Max:plasmid.