γ tubulin

Detailed experimental procedures have been described previously [4 (link)]. Briefly, whole cell lysates were prepared by resuspending cell pellets in cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin). Protein concentration was determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). Protein extract was loaded onto an SDS-polyacrylamide gel, separated by electrophoresis and then transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then incubated with primary antibodies against AMPKα, phospho-AMPKα (Thr172) and phospho-ACC (Ser79) (Cell signaling, Technology Inc, Beverly, MA) and γ-tubulin (Sigma-Aldrich, St Louis, MO) overnight at 4°C. After washing with TBS-T, the membrane was incubated with rabbit IgG secondary antibodies, and the signals were visualized using the ECL western blotting system (Millipore, Billerica, MA).

Unless specified otherwise, all chemicals were obtained from Sigma-Aldrich. Antibodies were: acetylated α-tubulin (T7451, Sigma-Aldrich, and D20G3; Cell Signaling Technology), ADP-ribosylation factor-like 13B (Arl-13B, NeuroMab clone N295B/66), Glutamylated tubulin (AB3201, Chemicon), Myelin basic protein (MBP, Aves Labs), γ-tubulin (T6557, Sigma-Aldrich), Histone-3P (06–570, Millipore), BrdU (M0744, DAKO) and PDGF-Rα (SC-338, Santa Cruz Biotech).
This study was carried out strictly in accordance with animal use guidelines of the National Fund for Science and Technology (FONDECYT) and with the Guide for the Care and Use of Laboratory Animals issued by the Institute for Laboratory Animal Research of the National Research Council (USA; National Academies Press, 2011). All procedures were approved by the Ethics Committee of the Faculty of Sciences, the University of Chile (certificates issued on July 31st, 2012 and October 4th, 2013) and were reviewed by the Bioethics Committee of the National Fund for Science and Technology (FONDECYT). Animals were neonatal (P1) Sprague Dawley rats obtained from the central animal housing facilities at the Catholic University of Chile. Suffering of animals was kept to a minimum and no procedures inflicting pain were performed.

Between 120.000 and 150.000 cells were grown in 24-well plates. The day after cells were treated or not with 15 µM RO-3306 for 18 h at 37 °C, 5% CO₂. Cells were washed with PBS, fixed with 4% PFA for 10 min at RT and permeabilized with PBS / 0.1% Triton X-100 for 5 min at RT. Blocking (RT, 20 min) and incubations with antibodies (RT, 1 h) were performed with 10% FBS in PBS 0.1% / Triton X-100 and washes were done with PBS 0.1% / Triton X-100 at RT for 3 × 5 min. The antibodies targeted α-tubulin (T9026, Sigma) and γ-tubulin (T6557, Sigma). An Alexa 555 Goat anti-Mouse antibody (A-21424, Invitrogen) was used as a secondary antibody. Nuclei were counterstained with 1 μg/mL DAPI for 2 min at RT and cells were mounted using the ProLong Gold antifade reagent (P10144, Thermofisher). Confocal microscopy pictures were taken with a Leica STELLARIS microscope. For counting lagging chromosomes, DNA bridges, multipolar mitosis or centrosome numbers, at least 200 cells were analyzed by eye for each condition.

Nuclear protein extracts were prepared using the Nuclear/Cytosol Fractionation kit (BioVision) following protocols provided by the manufacturer. Total lysates were prepared using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors and micrococcal nuclease). In the case of tissue extracts, lysates were homogenized using a Precellys 24 tissue homogenizer. For immunoblotting, protein extracts were resolved using NuPAGE 4–12% gradient Bis-Tris gels, transferred to nitrocellulose and incubated with the corresponding antibodies. For murine NANOG detection, we used Calbiochem, SC1000. For human NANOG detection, we used Cell Signaling, D73G4XP. Other antibodies used were: γ-TUBULIN (Sigma, GTU-88), phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling, 9101), total ERK1/2 (Cell Signaling, 9102), phospho-AKT (Ser473) (Cell Signaling, 4058), p63 (Novus Biologicals, 4A4, NB100-691) and SMC1 (Bethyl, A300-055A). Quantification of immunoblots was done using the ImageJ software. Uncropped images of the immunoblots are shown in Supplementary Figure S6.