Complete proteinase inhibitor cocktail tablets

After dissection, tissues were kept at –80 °C till use. To prepare protein lysates, tissues were homogenized with an Ultra-Turrax T10 (VWR) in 10 volumes (w/v) of ice-cold modified RIPA buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) supplied with Complete Proteinase Inhibitor Cocktail tablets (1873580, Sigma-Aldrich) and PhosSTOP phosphatase Inhibitor Cocktail tablets (0490683701, Sigma-Aldrich). After a further 5-min sonication step in an ultrasonic bath for shearing genomic DNA, the lysates were centrifuged at 16,200× g and 4 °C for 20 min to obtain the soluble protein.
Western blot analyses were performed as described earlier [44 (link)]. Primary antibodies were diluted in TBS-T (TBS with 0.1% Tween 20) and the respective dilution factors are summarized in Table A1. For total protein detection, membranes were incubated with SYPRO Ruby Protein Blot Stain (Thermo Fisher Scientific) according to the manufacturer’s protocol, prior to blocking. For caspase-3 analyses, a commercial sample of cell extracts treated with cytochrome c served as positive control (9663, Cell signaling). All fluorescence signals were detected and quantified using the ODYSSEY FC Imaging System with Image Studio software version 4.0 (both LI-COR Biosciences, Bad Homburg, Germany).

Mice striatal tissues were homogenized in ice-cold 10 volumes w/v modified RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, 5 mM EDTA pH 8.0) with Complete Proteinase Inhibitor Cocktail tablets (Sigma-Aldrich, 1873580) with a mechanical homogenizer. After a further 5-min sonication step with a bath sonicator for shearing genomic DNA, the lysates were centrifuged at 16,200× g at 4 °C for 20 min to isolate the soluble protein. Protein samples were denatured in Lithium dodecyl sulfate (LDS) buffer (NP0007, Thermo Fisher, Darmstadt, Germany) containing 100 mM DTT and separated using NuPAGE Bis-Tris 12% gel (Thermo Fisher, NP0349BOX). Blots were incubated overnight at 4 °C with the following primary antibodies: Anti-pro-brain-derived neurotrophic factor (BDNF) (1:500, Sigma-Aldrich, P1374-200UL), anti-nerve growth factor (NGF) (1:1000 Abcam, ab6199), anti-DARPP-32 (1:5000, Epitomics, 1710-1), anti-tyrosine hydroxylase (TH) at a concentration of 1:1000 (Merck Millipore, AB1542), anti-Iba1 at a concentration of 1:1000 (Wako, 019-1974), and anti-beta actin (1: 5000, Sigma-Aldrich, A5441). Florescence-conjugated secondary antibodies, anti-rabbit and anti-mouse at a dilution of 1:10000 (Li-COR Bioscience, 926-32211 and 926-68070), were used to detect the signals utilizing Li-COR Odyssey imaging system (Li-COR Bioscience).