Inducible Expression of SAS-6 Domains

Human telomerase-immortalized retinal pigment epithelial cells (hTERT-RPE1 or RPE1) were cultured in DME/F-12 (1:1) medium supplemented with 10% FBS and 1% penicillin-streptomycin. The expression constructs of the various SAS-6 DMs under the control of tetracycline-inducible promoter were made using the lentiviral vector pLVX-Tight-Puro vector (Clonetech) as described previously (Fong et al., 2014 (link)). Antibodies used in this study include anti-α-tubulin (Sigma-Aldrich), anti-HA.11 (Covance), anti-polyglutamylated tubulin (GT-335; Adipogen), anti-pericentrin and CPAP (Proteintech), anti-centrin2 (Millipore), anti-BrdU (AbD Serotec), CEP164 (Novus Biologicals), anti-STIL (Bethyl laboratories, Inc), anti–p53 and hSAS-6 (Santa Cruz Biotechnology; sc-6243, sc-376836 and sc-81431), and anti-CEP135 (abcam, ab75005). The antibody for N-terminal SAS-6 (sc-376836) is a monoclonal antibody raised against amino acids 1–300, with the actual epitope being mapped within amino acids 173–300 (Figure 1—figure supplement 1D). The antibody for C-terminal SAS-6 (sc-81431) is a monoclonal antibody raised against amino acids 404–657, with the epitope being mapped within amino acids 519–657 (Figure 1F).