Anti stil

Cells were lysed in lysis buffer [50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 5 mmol/L EGTA pH 8.0, 5 mmol/L EDTA pH 8.0, 1% (v/v) NP40, 0.1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, phosphatase inhibitor cocktail (25 mmol/L β-glycerophosphate, 50 mmol/L NaF, 5 mmol/L Na₂MoO₄, 0.2 mmol/L Na₃VO₄, 5 mmol/L EDTA, 0.5 μm microcystin], complete EDTA-free protease inhibitor cocktail (Roche). A total of 8% or 10% SDS polyacrylamide gels were prepared and proteins were separated followed by the transfer to nitrocellulose membranes. The following antibodies were used: anti-α-tubulin (B-5-1-2, 1:1,000, Santa Cruz Biotechnology, #sc-23948, RRID:AB_628410), anti-β-actin (AC-15, 1:10,000, Sigma-Aldrich, #A5441, RRID:AB_476744), anti-chTOG (H-4, 1:400, Santa Cruz Biotechnology, #sc-374394, RRID:AB_10987687), anti-Plk4 (6H5, 1:400, Merck Millipore, #MABC544, RRID:AB_2893410), anti-STIL (1:5,000, Bethyl Laboratories, #A302442A, RRID:AB_1944269), anti-HER2 (1:1,000, Cell Signaling Technology, #2242, RRID:AB_331015), anti-mouse/rabbit-HRP (1:10,000, Jackson ImmunoResearch, #115-035-146, #111-035-144, RID:AB_2307392). All Western blots were performed from at least three independent experiments.

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).