T6557

Cells were fixed with 1.5% formaldehyde in PBS for 5 min and post-fixed with ice-cold methanol for 5 min at −20 °C. After several washes in PBS, the cells were blocked in 1% BSA (Sigma) and 0.05% Tween-20 (Sigma) in PBS for 15–0 min and then incubated with primary antibodies. Alexa Fluor 555-, 405-, 488- or 647-conjugated secondary antibodies (Invitrogen) were used to visualize the proteins. DNA was stained with Hoechst 33342 (Invitrogen). The following primary antibodies were used for immunostaining in this study: mouse monoclonal anti-Sas6 (1:500; sc-81431; Santa Cruz), mouse monoclonal anti-γ-tubulin (1:5,000; T6557; Sigma), mouse anti-centrobin (1:600; ab70448; Abcam), rabbit anti-Cep135 (1:500; ab75005; Abcam), rabbit anti-Cep152 (1:4,000; A302-479 A; Bethyl), rabbit anti-γ-tubulin (1:5,000; T3559; Sigma), rabbit anti-Cep215 (1:2,000; 06-1398; Millipore), goat anti-CPAP (1:500; sc-66747; Santacruz), rabbit anti-CPAP (1:500; 11517-1-AP; Proteintech), mouse anti-GT335 (1:2,000; AG-20B-0020-C100; Adipogen), mouse anti-Plk1(pT210) (1:500, 558400; BD Biosciences) rabbit anti-Plk4 (1:100; kind gift from Dr Kyung S. Lee, NIH Bethesda, USA), rabbit anti-hPOC5 (1:1,000; a kind gift from J. Azimzadeh, Institute Jacques Monod, Paris, France), rabbit anti-Cep120 (1:1,000; a kind gift from Moe R Mahjoub, Washington University in St Louis, USA).

Cells on coverslips were fixed with 4% paraformaldehyde/2% Sucrose for 15 min, and triton extracted (0.5% Triton X-100 in PBS) for 4 min. Cells were blocked with 5%BSA/PBST and then incubated with respective antibodies for 30 min at 37 °C followed by incubation with secondary antibodies (FITC or Rhodamine) for 30 min at 37 °C. Primary antibodies used in immunofluorescence studies were BRCA1 (Upstate; 1:500), phospho-53BP1(S1778) (Cell Signaling, 2675S; 1:200), RPA (Cal Biochem, NA13; 1:100), 53BP1 (Bethyl Labs, A300-272A; 1:2,000), Rad51(Santa Cruz, SC-8349; 1:150), Mre11 (Genetex, GTX70212 1:200), CtIP (generous gift from Dr. Richard Baer), HA (Covance, MMS-101P; 1:500) and γ-H2AX (Millipore, 05-636; 1:5,000). For TPX2 (Bethyl Labs, A300-429A; 1:400) and γ-tubulin (Sigma- Aldrich, T6557; 1:1,000) staining, the cells were pre-fixed with acetone:methanol (3:7) at −20 °C for 10 min, followed by triton extraction (0.2% triton-X-100 in 20 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose) at room temperature. Primary and secondary antibody staining was carried out as described above.

A rabbit polyclonal antibody against APP (1:1000) (A8717, Sigma), rabbit polyclonal anti‐Nav1.6 (1:200) (ASC‐009, Alomone Laboratories), rabbit polyclonal anti‐Nav1.2 (1:200) (ASC‐002, Alomone Laboratories), BACE1 (1:1000) (ab2077, Abcam), Phospho‐NFAT1 (1:500) (AF8011, Affinity Biosciences), NFAT1 (1:500) (DF7189, Affinity Biosciences), β‐actin (1:2000) (ab8227, Abcam), and γ‐tubulin (1:2000) (T6557; Sigma) were obtained from the respective commercial sources. TTX (1 µM, Sigma), EGTA (0.5 mM, sigma), Aβ1‐42 (5 µM, A‐1163–2, rPeptide), and KB‐R7943 (5 µM, MCE) were purchased.

Commercial antibodies used were as follows: anti-Acetylated tubulin (^Actub, clone 6-11B-1, 1/10000, T6793, Sigma), anti-Gamma-tubulin (GTU-88, 1/1000, T6557, Sigma), anti-b-actin (clone AC-15, 1/30000, A5441, Sigma), anti-PACSIN1 (1/100, 196 003, Synaptic Systems), anti-PACSIN2 (1/250, ab37615, Abcam), anti-PACSIN2 (1/500, 10518-2-AP, Proteintech), anti-PACSIN3 (1/100, ab37612, Abcam), anti-Pericentrin (PCTN, 1/5000, NB100-61071, Novus Biologicals), anti-EHD1 (EPR4954, 1/500, ab109311, Novus Biologicals), anti-RPGRIP1L (1/200, 55160-1-AP, Proteintech), anti-TMEM67 (1/200, 12780-1-AP, Proteintech), anti-CEP164 (1/500, sc-240226, Santa Cruz), anti-CP110 (1/1000, 12780-1-AP, Proteintech), anti-CEP97 (1/1000, A301-945A, Bethyl), anti-Arl13b (1/300, clone N295B/66, NeuroMab/UC Davis), anti-GFP Alexa 488 (1/1000, A21311, Molecular Probes Life Technologies), Phalloidin conjugated with Alexa 488 (1/50, A12379, Molecular Probes Life Technologies), Hoechst (1/3000, H3570, Molecular Probes Life Technologies) and all secondary antibodies were from Life Technologies. The rabbit anti-RAB8A antibody was a gift from Johan Peränen (University Helsinki, Finland). SNAP-Cell647-SiR reagent was purchased from New England Biolabs. Nocodazole (Calbiochem) was from Sigma. Doxycyclin hydrochloride was obtained from Sigma and used according to manufacturer’s instruction.

For transient transfection, 293 T cells were co-transfected with an rtTA plasmid and the Lenti.TRE-PPARαΔ13_PGK-Cre construct using LF2000 (ThermoFisher Scientific), and were treated (or not) with 2 μg ml⁻¹ doxycycline one day later for 24 hr to monitor PPARαΔ13 induction. For all experiments, cells were lysed in RIPA (150 mM NaCl, 20 mM Tris pH 7.4, 0.1% SDS, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, one protease inhibitor cocktail tablet (11836145001, Roche)). Proteins were quantified using a BCA assay, and 50 μg (or 5 μg after Seahorse) was used for western blot. Antibodies used are anti-PPARα (PA1-822A, ThermoFisher Scientific, 1:1000; RRID:AB_2165595), anti-GLUT1 (07–1401, Millipore, 1:1000; RRID:AB_1587074), anti-GLUT3 (ab41525, Abcam, 1:1000; RRID:AB_732609), anti-β-tubulin (sc-9104, Santa Cruz Biotechnology, 1:1000; RRID:AB_2241191), and anti-γ-tubulin (T6557, Sigma-Aldrich, 1:10’000; RRID:AB_477584).

Primary antibodies used were mouse-anti-NDPK-B (MC-412, Kamiya, Seattle, WA, USA, 1:1000), rabbit-anti-NDPK-A (sc-343, Santa Cruz, Heidelberg, Germany, 1:500; detects both NDPK-A and NDPK-B in mouse retinae), mouse-anti-Ang-2 (sc-74403, Santa Cruz, Heidelberg, Germany, 1:500), mouse-anti-O-GlcNAc (ab-2739, Abcam, Cambridge, UK, 1:1000), rabbit-anti-OGA (SAB4200267, Sigma, Munich, Germany, 1:1000), rabbit-anti-OGT (O-6264, Sigma, Munich, Germany, 1:1000), rabbit-anti-N1-phosphohistidine (MABS1330, Millipore, Darmstadt, Germany, 1:1000), mouse-anti-γ-tubulin (T6557, Sigma-Aldrich, Munich, Germany, 1:2000), rabbit-anti-GFAT (obtained in cooperation with Weigert, Tübingen), and sheep-anti-pGFAT (MRC-PPU s343c) for immunoblotting. The secondary antibodies used were rabbit anti-mouse peroxidase (A9044, Sigma-Aldrich, Munich, Germany, 1:20,000), goat-anti-rabbit peroxidase (A9169, Sigma-Aldrich, Munich, Germany, 1:40,000), and donkey-anti-sheep peroxidase (Sigma-Aldrich). qPCR primers were obtained from Applied Biosystems, ThermoFischer: GFAT Hs00899865_m1, OGA Hs00201970_m1, OGT Hs00269228_m1, and 18S Hs03003631_g1. Gelatin from porcine skin (48720, Fluka, Bucharest, Romania) was used as a 1% solution in PBS. Thiamet G (TMG; SML0244, Sigma-Aldrich, Germany) treatment was given at 10 µM for 24 h.

Cells were seeded on coverslips, treated, fixed for 20 min with 2% paraformaldehyde/ 2% sucrose and permeabilized for 15 min with 0.1% Triton-X 100. Following 1 h blocking with 2.5% donkey serum in 0.05% PBS/Tween, coverslips were incubated as needed with primary antibodies: γH2AX S139 (1:1500, #05–636-I, Millipore), 53BP1 (1:1500, #sc-22760, Santa Cruz Biotechnology), cyclin A (1:1000, #GTX-634–420, GeneTex) or Phalloidin (1:50, #A12379, Invitrogen). For BrdU staining (1:500, #RPN20AB, GE Healthcare), cells were fixed with ice-cold methanol (40 s) and acetone (20 s), followed by DNA denaturing in 1.5 N HCl for 40 min. For staining of centrosomes (1:1000, #T6557, Sigma-Aldrich) and microtubules (1:1000, #T9026, Sigma-Aldrich), cells were fixed for 10 min with ice-cold methanol, followed by hydration with PBS. Following 1 h of incubation with primary antibodies, cells were washed (3 x/10 min each) with 0.05% PBS/Tween, incubated for 1 h with anti-donkey Alexa 488 or 546 (1:200, Invitrogen), washed, stained with DAPI (#10236276001, Roche) and mounted on slides with Mowiol (Sigma-Aldrich). Slides were analyzed with ×40 or100 x objectives using an Axio Observer microscope (Zeiss).