Goat anti actin 1 19

Cells were lysed on ice in 20 mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100, 150 mM NaC1, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄), and complete protease inhibitors (Roche). After centrifugation, the supernatants were mixed with an equal volume of 2 × sample buffer (125 mM Tris-HCl, 0.01% bromphenacyl bromide, 4% SDS, 20% glycerol and 200 μM dithiothreitol). Samples were boiled for 5 min and analysed by immunoblotting. The following antibodies were used: goat anti-actin (I-19; 1:1,000 dilution; Santa Cruz), mouse anti-GFP (B-2; 1:1,000 dilution; Santa Cruz), and rat anti-HA (3F10; 1:500 dilution; Roche). Before assays, HEK293 cells stably expressing both pCI-GFP-mature Aire and pTRE2hyg-HA-immature Aire or pTRE2hyg were treated with doxycycline (Dox; 10 μg ml^–1; Clonetech) in the presence or absence of MG132 (10 μM; Sigma-Aldrich) for specified times. The signals were measured using ImageJ public-domain software (imagej.nih.gov/ij/) and standardized to β-actin. In Fig. 5c,d, images have been cropped for presentation. Full size images are presented in Supplementary Figure 9.

Cells were harvested and lysed in ELB buffer (150 mM NaCl; 50 mM Hepes, pH 7.5; 5 mM EDTA; 0.1% NP-40) containing protease inhibitors (Complete; Roche) for 30 min. Protein concentrations were determined using the BCA protein assay kit (Pierce). Primary antibody used to detect mouse RECQL was rabbit polyclonal anti-Recql (A300-450A; 1/1,000; Bethyl Laboratories). For detection of human RECQL, mouse monoclonal anti-Recql (A-9, Sc166388; 1/250; Santa Cruz) was used. For detection of actin, polyclonal goat anti-actin (I-19; 1/1,000; Santa Cruz) was used. Secondary antibodies used were IR Dye 800CW donkey antigoat IgG (LI-COR) and IR Dye 800CW goat-antimouse IgG (LI-COR).

Cells were lysed in Tris–HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM, and protease inhibitors. Lysates were separated on an 8% acrylamide gel and immunoblotted using standard procedures. Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk and incubated overnight at 4 °C with the following antibodies: rabbit anti-Foxm1 (Santa Cruz Biotechnology, sc-502), rabbit anti-Hsp70, (Santa Cruz Biotechnology, sc-33575), mouse anti- βIIItub (MAB 1637 Millipore), goat anti-actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-Nanog (PA1–41577, Thermo Fisher), anti-α-Tubulin Antibody (T9026 SIGMA). HRP-conjugated secondary antisera (Santa Cruz Biotechnology) were applied and binding visualized by enhanced chemiluminescence (ECL Amersham).

HEK293T, human prostate adenocarcinoma LNCaP, Du145 and PC3 cells lines were purchased from the American Type Culture Collection, Manassas, VA, USA. The Docetaxel resistant cell lines Du145 and PC3 were developed as previously described [31 (link)]. HEK293T were cultured in DMEM (BioWest, Nuaillé, France), Docetaxel sentitive and resistant Du145 cells and LNCaP cells were cultured in RPMI-1640 (BioWest) and PC3 DS and PC3 DR in F12K nutrient mixture medium (Thermo Fisher Scientific, Waltham, MA, USA). Culture media were supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 0.1 mM non-essential amino acids (all from BioWest). Docetaxel resistant cell lines were maintained with 2.5 nM of Docetaxel (Sigma-Aldrich, St. Louis, MO). Antibody to PTOV1 was produced and purified as previously described [8 (link), 9 (link)]. Additional antibodies were obtained from: goat anti-actin (I-19) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-HA.11 (16B12) (Covance, MA); mouse anti-Cyclin B1(V152) (Abcam, Cambridge, MA); rabbit anti-PARP1 (H-250) (Santa Cruz Biotechnology). Lentiviral vectors carrying short-hairpin RNA (shRNA, TRCN0000143905, TRCN0000140104 and TRCN0000139737) to PTOV1 were obtained from Sigma.

The global protein synthesis ability of parental cells and G2 spheres was evaluated by puromycin immunodetection using the SUnSET assay^{48 (link)}. Cells (3 × 10⁵) were seeded in 6 well plates and 24 h later were treated with 10 μg/ml puromycin for 10 min. Non-treated cells were used as negative control; cells treated with 10 μg/ml puromycin and 10 μg/ml cycloheximide for 10 min were used as positive control. Cell lysates were collected and analyzed by Western blotting as previously described^{42 (link)}. Antibodies used were goat anti-actin (I-19) (Santa Cruz Biotechnology) and Mouse Puromycin (12D10) (Millipore).