Cycloheximide (chx)

The nucleus accumbens lysates from the same drug treatment experiment (W and O1 or O2) were analyzed in each polysomal ultracentrifugation experiment. An equal volume of nucleus accumbens lysate (generally 300 μl) was layered onto a 15–45 % (w/v) sucrose gradient containing 100 μg/ml cycloheximide (VWR) and centrifuged in a Beckman SW41Ti rotor at 38,000 rpm at 4 °C for 2 h. Gradients were collected in 1-ml fractions with continuous monitoring of absorbance at 254 nm using an Isco syringe pump with UV-6 detector (Teledyne Isco Inc.). Samples were stored at −80 °C until further use. The polysomal analysis was repeated three times using lysates from different drug administration experiments.

HEK293T cells (∼75% confluent) were transfected with 0.8 μg of pCB328 per 35-mm well using BioT according to the manufacturer's protocol. At 24 h post-transfection, cells were pulse labeled for 10 min with 0.1 mCi/mL ³⁵S-labeled methionine (MP Biomedicals) in DMEM lacking Met and Cys. Labeling was quenched by the addition of 0.1 mg/mL cycloheximide (VWR) in complete DMEM containing Met and Cys. Denaturing immunoprecipitation was then carried out according to the Tansey protocol (Tansey, 2007 ). Briefly, cells were immediately lysed by rapid scraping in 150 μL of TSD buffer (50 mM Tris-HCl [pH 7.4], 1% SDS, 5 mM DTT) and snap-freezing in liquid nitrogen. Samples were then heated at 95°C for 10 min and diluted with 10 volumes of TNN buffer (0.5% NP-40, 0.25 M NaCl, 5 mM EDTA, 50 mM Tris-HCl [pH 7.4]) containing the complete protease inhibitor mixture (Roche). Samples were then immunoprecipitated by the addition of 5 μl of anti-FLAG-M2 magnetic beads (Sigma) and incubation with rocking at 4°C for 3 h. Immunoprecipitated proteins were then washed 3 times in TNN buffer and once in phosphate-buffered saline, followed by resuspension in 20 μl of SDS sample buffer, heating at 95°C for 10 min, 4 to 15% SDS-PAGE, and autoradiography.

Trypsin-EDTA (Cat# 25-053-CI), PBS (Cat# 21-031-CV), and laminin (Cat# 354232) were purchased from Corning. Recombinant EGF was purchased from Life Technologies (Cat# PHG0311). IGF was purchased from Sigma-Aldrich (Cat# I3769). PIM447 was purchased from Selleck Chemicals (Cat# S7985). AZD1208 was acquired from AdooQ Bioscience (Cat# A13203-750). DMSO was purchased from Thermo Fisher Scientific (Cat# 97064-724). Cycloheximide was purchased from VWR (Cat# 97064-724), and doxycycline was purchased from Sigma-Aldrich (Cat# D9891-5G). Recombinant myelin basic protein (MBP) was a gift from Dr. Greg Rogers, and recombinant ABI2 protein was purchased from Origene (Cat# TP300637). Radio-labeled ATP was purchased from Perkin Elmer (Cat# BLU502A). The antibody to ABI2 was purchased from Bethyl Laboratories (Cat# A302-499A-M). GFP (Cat# 2956S), HA (Cat# 3724S), HIF-1a (Cat# 14179S), p-IRS1 [S1101] (Cat# 2385S), PIM1 (Cat# 3247S), and WAVE2 (Cat# 3659S) antibodies were purchased from Cell Signaling Technology. The antibody for actin was purchased from BD Biosciences (Cat# 61656). PIM1 (Cat# ab75776) and Ki67 (Cat#ab833) antibodies used for immunohistochemistry were purchased from Abcam.

HEK293T cells (∼75% confluent) were transfected with 0.8 μg plasmid per 35-mm well using BioT according to the manufacturer's protocol. At 24 h posttransfection, cells were pulse labeled for 15 min with 0.1 mCi/ml ³⁵S-labeled l-methionine (MP Biomedicals) in DMEM lacking Met and Cys. Labeling was quenched by the addition of 0.1 mg/ml cycloheximide (VWR) in complete DMEM containing Met and Cys. Cells contained in an entire 35-mm well were lysed at the indicated time points of a chase by rapid scraping in 150 μl of TSD buffer (50 mM Tris-HCl [pH 7.4], 1% SDS, 5 mM DTT) and snap-freezing in liquid nitrogen. Samples were then heated at 95°C for 10 min and diluted with 10 volumes of TNN buffer (0.5% NP-40, 0.25 M NaCl, 5 mM EDTA, 50 mM Tris-HCl [pH 7.4]) containing the complete protease inhibitor mixture (Roche). Total ³⁵S (in the 10% CCl₃COOH-insoluble fraction) in samples was then measured, and volumes were adjusted to equalize ³⁵S among different samples. Normalized samples were then immunoprecipitated by the addition of 5 μl of anti-FLAG-M2 magnetic beads (Sigma) and incubation with rocking at 4°C for 3 h. Immunoprecipitated proteins were then washed 3 times in TNN buffer and once in phosphate-buffered saline, followed by resuspension in 20 μl of SDS sample buffer, heating at 95°C for 10 min, 4 to 15% SDS-PAGE, and autoradiography.

BSR-T7 cells were seeded in 6-well plates and incubated 1 day later in phosphate-free DMEM (Gibco; catalog no. 11971-025) for 30 min followed by a 30-min incubation in phosphate-free DMEM containing 10 μg/ml actinomycin D (Sigma; catalog no. A5156) and 100 μg/ml cycloheximide (VWR; catalog no. 94271). Cells were then infected for 30 min with sucrose cushion-purified virus at an MOI of 100. Virus solutions were replaced by 1 ml phosphate-free DMEM containing 10 μg/ml actinomycin D, 100 μg/ml cycloheximide, and 10 μl of phosphorus-32 radionuclide (PerkinElmer; catalog no. NEX053H005MC). At 2, 3, 4, 5, and 6 h postinfection, RNA was extracted using TRIzol reagent (Invitrogen; catalog no. 15596018) following the manufacturer’s protocol. RNA was boiled at 100°C for 1 min, incubated on ice for 2 min, mixed with a 1.33× loading buffer (33.3 mM citrate pH 3, 8 M urea, 20% sucrose, and 0.001% bromophenol blue), and analyzed on a 25 mM citrate, pH 3, 1.75% agarose, 6 M urea gel run for 18 h at 4°C and 180 V. Gels were fixed (in 30% methanol and 10% acetic acid), dried, and exposed overnight to a phosphor screen (GE Healthcare), and the radiolabeled RNA products were visualized using a Typhoon FLA 9500 scanner (GE Healthcare).