Cycloheximide (chx)

Yeast cells growing exponentially were treated or not with auxin for 20 min. 50 µg/mL cycloheximide (Sigma) was added directly to the culture medium. Cells were collected by centrifugation, rinsed with buffer K [20 mM Tris-HCl pH 7.4, 50 mM KCl, 10 mM MgCl₂] supplemented with 50 µg/mL cycloheximide and collected again by centrifugation. Dry pellets were resuspended with approximately one volume of ice-cold buffer K supplemented with 1 mM DTT, 1 × Complete EDTA-free protease inhibitor cocktail (Roche), 0.1 U/µL RNasin (Promega) and 50 µg/mL cycloheximide. About 250 µL of ice-cold glass beads (Sigma) were added to 500 µL aliquots of the resuspended cells and cells were broken by vigorous shaking, three times 2 min, separated by 2 min incubations on ice. Extracts were clarified through two successive centrifugations at 13,000 rpm and 4°C for 5 min and quantified by measuring absorbance at 260 nm. About 30 A₂₆₀ units were loaded onto 10–50% sucrose gradients in buffer K, and then centrifuged for 150 min at 39,000 rpm and 4°C in an Optima L-100XP Ultracentrifuge (Beckman-Coulter) using a SW41Ti rotor without brake. Following centrifugation, 18 fractions of 500 µl each were collected from the top of the gradients with a Foxy Jr. apparatus (Teledyne ISCO). The absorbance at 254 nm was measured during collection with a UA-6 device (Teledyne ISCO).

This was performed as described^{51 (link)}. Briefly, fully differentiated brown adipocytes on a 15-cm plate were treated with or without Forskolin or CL-316243 for 9 h. The cells were treated with 100 µg ml⁻¹ cycloheximide (Sigma, Cat#C7698) for 5 min, harvested in 5 ml ice-cold PBS containing 100 µg ml⁻¹ cycloheximide, and centrifuged at 4 °C. 425 µl of hypotonic buffer (5 mM Tirs-HCl, pH 7.5; 2.5 mM MgCl₂; 1.5 mM KCl and protease inhibitor cocktail, EDTA-free), 5 µl of 10 mg ml⁻¹ cycloheximide, 1 µl of 1 M DTT and 100 units of RNasin (Promega, Cat#N251A) were added into cell pellet and vortexed for 5 s, and then 25 µl of 10% TritonX-100 and 25 µl of 10% sodium deoxycholate were added and vortexed for 5 s. Samples were centrifuged at 4 °C, and OD₂₆₀ of supernatants was measured using NanoDrop. 18 OD₂₆₀ units were loaded onto sucrose gradient (10–50%) and centrifuged at 35,000 rpm with a SW40 rotor for 2 h at 4 °C. Each sucrose gradient fraction was collected and RNA was isolated for RT-qPCR analysis. Levels of mRNA in polysome fractions were normalized with the sum of mRNA levels in all the fractions.

Ribosomes were pelleted as described previously (93 (link)). Briefly, C57BL/6 MEFs were plated on 10-cm-diameter plates at 3 × 10⁵ cells per plate. The next day, the cells (∼90% confluent) were infected with MAV-1 at an MOI of 5. C57BL/6 MEF lysates were collected at 48 hpi by scraping the cells in ice-cold PBS containing 100 µg/ml cycloheximide (Sigma C7698), pelleting, and resuspending in lysis buffer, which contained 10 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 4 mM DTT, 0.5% NP-40, 100 µg/ml cycloheximide, 20 U/ml RNasin (Promega catalog no. N2511), 10% sucrose, and 1× protease inhibitors (protease inhibitor cocktail kit; Thermo Scientific catalog no. 78410). Cells were lysed by passage through a chilled 26-gauge needle five times and were cleared by centrifugation for 10 min at 21,000 × g at 4°C. A 400-µl volume of cleared lysate (optical density at 260 nm [OD₂₆₀] of 10) was layered onto 25% sucrose and centrifuged at 29,500 rpm in an SW41 rotor (average relative centrifugal force [rcf], 107,458) for 4 h at 4°C. After pelleting, the supernatant was removed by the use of a micropipette, and 350 µl of Buffer RLT Plus (from Qiagen RNeasy minikit) (4°C) was added to the pellet for collection of the RNA. The RNA was purified immediately using an Qiagen RNeasy minikit (Qiagen catalog no. 74134) and stored at −80˚C until analysis.

Polyribosomes were generated as described before [20] (link), [28] (link) with minor modifications. 40×10⁶ of I/11 cells were incubated with 0.1 mg/ml cycloheximide (Sigma Aldrich) for 10 min at 37°C, washed with cold PBS containing 0.1 mg/ml cycloheximide and lysed in buffer containing 10 mM Tris-HCl, 12 mM MgCl₂, 140 mM NaCl, 0.5% Nonidet-P40, 500 U/ml RNAsin (Promega), 0.1 mg/ml cycloheximide, 20 mM dithiothreitol and SigmaFast protease inhibitor (Sigma Aldrich). After removal of nuclei (3000×g; 3 min) heparin was added to 3.75 mg/ml and lysates were centrifuged at 13000×g for 13 min. Supernatants were loaded on 10.5 ml 7–46% linear sucrose gradients containing 10 mM Tris-HCl, 12 mM MgCl₂, 140 mM NaCl and centrifuged at 190,000×g for 3 hours. Measurement of absorbance at 254 nm, visualization and fractionation were done by Econo system (Biorad).

For degradation assays, cells were transfected with nanoluc or luciferase tagged CUL3, CUL3-∆9, KLHL3, or Flag-WNK4 constructs and 24 h after transfection, cells were treated with cycloheximide (20 μg/mL) (Sigma; Saint Quentin Falavier, France). Cells were collected at different time points after cycloheximide addition and then lysed in passive lysis buffer (Promega, Charbonnières les Bains, France), and luminescence was measured in presence of coelenterazine H or NanoGlo (Promega, Charbonnières les Bains, France) or lysed in IP lysis buffer and analyzed by Western blot as described above.
The apparent affinity of WT and ∆9 CUL3 for themselves or Bacurd, KLHL7, and KLHL3 was evaluated by BRET. In each experiment, a fixed amount of BRET donor plasmid (CUL3-, Bacurd-, KLHL7-luciferase) was transfected in HEK293 cells (6-well plates) in association with increasing amounts of the BRET acceptor (YFP-CUL3-WT or −∆9). Signals were measured using a Mithras LB 940 multimode reader (Berthold, Thoiry, France). BRET results were expressed in milli-BRET units (mBRET), or % of maximal BRET signal plotted as a function of YFP/Rluc ratio, in which YFP represents the actual amount of expressed BRET acceptor and Rluc the amount of BRET donor in each sample.