Complete mini edta free cocktail

Total proteins from mouse ventral midbrain, striatum and cerebellum samples were isolated in NP‐40 buffer (20 mM Tris–HCl pH 8; 137 mM NaCl; 10% glycerol; 1% NP‐40; 2 mM EDTA and protease inhibitors (cOmplete Mini EDTA‐free cocktail, Roche)) 1:20 (wt/vol). Protein concentration was determined using a bicinchoninic acid kit (Pierce, Rockford, IL). After boiling in Laemmli's buffer, 20 µg of protein was separated by electrophoresis on a 12% sodium dodecyl sulphate–polyacrylamide gel, transferred to nitrocellulose membrane, and blocked with 2% BSA, 5% or 2% non‐fat dried milk in PBS containing 0.05% Tween‐20 (vol/vol). Overnight incubation with primary antibody at 4°C followed. Primary antibodies were rat anti‐MHC II (1:750; eBioscience), mouse anti‐interferon‐γ (1:500; ThermoScientific, Cambridge, UK), rabbit anti‐tumour necrosis factor‐α (1:200; Abcam, Cambridge, UK), rabbit anti‐interleukin‐1β (1:500; Abcam), and mouse anti‐β‐actin (1:25,000; SigmaAldrich). Blots were then washed a second time in PBS‐Tween (0.05%) and incubated with an appropriate secondary antibody (Jackson Immuno Research). Blots were washed in PBS‐Tween (0.05%) and developed using the Supersignal West Dura kit (Pierce) as per manufacturer's instructions. Bands were visualized with an AlphaInnotech digital imaging system (San Leandro, CA) and quantified with AlphaEase FC 5.02 software.

COS-7 or COV434 cells were lysed 48 h after transfection in 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (pH 7.6) supplemented with protease inhibitors PMSF 1 mM and Complete mini EDTA-free cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitors (Sigma Aldrich, St Louis, MO, USA). Clarified lysates were used for Western blotting or immunoprecipitated using Anti-V5 Agarose Affinity Gel (Sigma Aldrich, St Louis, MO, USA) according to instructions. Precipitated proteins were eluted in 2x-SDS sample buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol and 0.1% bromophenol blue). 100 mM DTT was further added to the samples. Eluates were separated by SDS-PAGE using NuPage Bis-Tris 4–12% Gels (Invitrogen Corporation) and proteins were electrotransfered onto PVDF membranes (Hybond-P, GE Healthcare, Waukesha, WI, USA). Antibodies used for detection were: α-GATA4 1∶1000 (sc-1237, Santa Cruz Biotechnology,), α-FOXL2 1∶1000 (IMG-3228, Imgenex) and α-SMAD3 1∶1000 (#51–1500, Invitrogen Corporation).

Tetracycline-inducible U2OS (U2OS-TR) cells and tetracycline-inducible U2OS cells stably transfected with pcDNA4/TO/myc-K294DNLS + NES-Flag (U2OS-K294A) were described previously [8 (link)]. Cells were maintained in a humidified incubator at 37°C under 5% CO₂ and were discarded after no more than 10 passages. Cells were grown in McCoy's 5A medium (Thermo Fisher 116600) supplemented with 10% tetracycline-free fetal bovine serum (FBS, Atlanta Biologicals S10350). Triplicate cultures of parental U2OS-TR or K294A-expressing cells at 70–80% confluence were switched to medium without or with 1 µg ml⁻¹ of doxycycline for 24 h. Prior to harvest cultures were washed three times with phosphate-buffered saline (PBS) and lysed using ice-cold lysis buffer (0.1 M HEPES, pH 8.5, 6M guanidine hydrochloride supplemented with one tablet of protease inhibitor (cOmplete Mini EDTA-free cocktail, Roche Life Science) and one tablet of phosphatase inhibitor (PhosSTOP, Roche Life Science). The cell lysates were sonicated using Sonic Dismembrator Model 100 (Fisher Scientific) for three cycles of alternating 30 s bursts followed by 30 s rest followed by centrifugation at 16 000× g for 15 min at 4°C. The protein concentration of the collected supernatant was determined by a bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific).