Pageruler plus prestained protein ladder

Western blots were performed according to standard protocols. Briefly, cell lysis was conducted with RIPA buffer (Bio-Rad formulation) supplemented with protease inhibitor cocktail (1:50; Sigma Aldrich, Vienna, Austria), followed by sonication (80% amplitude, 2 × 15 s). Samples were denatured in 1× Laemmli sample buffer and resolved on a 7.5 or 12.5% SDS-PAGE gel together with PageRuler™ Plus Prestained Protein Ladder (Fisher Scientific, Vienna Austria). Blots were blocked and antibodies diluted in 5% BSA (Sigma Aldrich) in TBS-T. The following antibodies were used: MICU1 (D4P8Q, 1:1000, Cell Signaling Technology, MA, USA), and Histone H3 (1B1B2, 1:1000, Cell Signaling Technology). HRP labeled anti-mouse (PI-2000, 1:1000, Vector Laboratories, Burlingame, USA) and anti-rabbit (sc-2357, 1:1000, Santa Cruz Biotechnologies) were used as secondary antibodies. For visualization, the SuperSignal West Pico PLUS kit (Fisher Scientific) was used and detection was conducted on the ChemiDoc System (Bio-Rad Laboratories, Vienna, Austria).

Glioblastoma cell lines and hCMEC/D3 cells were treated during 24 h in the absence of FBS and in the absence or the presence of UII (10^–9 M). Cell lysates (20 μg total proteins) were prepared in ice-cold Lysis Buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 0.1% SDS), mixed with Laemmli buffer and loaded onto 4–12% polyacrylamide gels (CliniSciences, Nanterre, France). Proteins were transferred onto PVDF membranes, blocked with 5% non-fat milk and 5% bovine serum albumin, and incubated with the anti-pre-pro UII (Sigma, HPA-01700), UT (H-90) (Santa-Cruz, sc-20940), or β-tubulin (Santa-Cruz, sc-9104) overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies (Santa-Cruz) for 2 h at room temperature. Immunoreactive bands were visualized by using the ECL Western blotting substrate (GE Healthcare, Aulnay-sous-Bois, France) and their molecular weight were determined by using PageRuler Plus prestained protein ladder (10–250 kDa) markers (Fisher Scientific).

Western blots were performed according to standard protocols. Briefly, cell lysis was conducted with RIPA buffer (+1% Triton-X100) (Bio-Rad formulation) supplemented with protease inhibitor cocktail (1:50; Sigma Aldrich, Vienna, Austria). Samples were frozen in liquid nitrogen and thawed for three times and incubated for 30 min on ice. Protein amounts were measured with Pierce BCA Protein Assay Kit (ThermoScientific, United States). 40 µg protein samples were loaded on a 12.5% SDS-PAGE gel together with PageRuler™ Plus Prestained Protein Ladder (Fisher Scientific, Vienna Austria). Blots were blocked (5% milk) and antibodies diluted in 5% milk in TBS-Tween. The following antibodies were used: Mitofusin-2 (D2D10, 1:1,000, Cell Signaling Technology, MA, United States), and β-Actin (D6A8, 1:1,000, Cell Signaling Technology). HRP labeled goat-anti-rabbit (sc-2054, 1:5,000, Santa Cruz Biotechnologies) was used as secondary antibody. For visualization, the SuperSignal West Pico PLUS kit (Fisher Scientific) was used and detection was conducted on a ChemiDoc System (Bio-Rad Laboratories, Vienna, Austria).

Western blots were performed according to standard protocols. Briefly, cell lysis was conducted with RIPA buffer (Bio-Rad formulation) supplemented with protease inhibitor cocktail (1:50; Sigma Aldrich, Vienna, Austria), followed by sonication (80% amplitude, 2 × 15 sec). Samples were denatured in 1 × Laemmli sample buffer and resolved on a 7.5% or 12.5% SDS-PAGE gel together with PageRuler™ Plus Prestained Protein Ladder (Fisher Scientific, Vienna Austria). Blots were blocked and antibodies diluted in 5% BSA (Sigma Aldrich) in TBS-T. The following antibodies were used: MCU (D2Z3B, 1:1000, Cell Signaling Technology, MA, USA), Opa1 (D6U6N, 1:1000, Cell Signaling Technology) and β-actin (sc-47778, 1:500, Santa Cruz Biotechnology, Heidelberg, Germany). HRP labeled anti-mouse (PI-2000, 1:1000, Vector Laboratories, Burlingame, USA) and anti-rabbit (sc-2357, 1:1000, Santa Cruz Biotechnologies) were used as secondary antibodies. For visualization, the SuperSignal West Pico PLUS kit (Fisher Scientific) was used and detection was conducted on the ChemiDoc System (Bio-Rad Laboratories, Vienna, Austria).

Immunoblots were carried out as described by our group [23 ,24 (link)]. Original immunoblots see File S1. Antibodies used for this assay were: BCL-XL (#ab32370), survivin (#ab134170), p21 (#ab109520), BAX (#ab32503), BAK (#ab32371), BIM (#ab32158), HDAC3 (#ab32369), GAPDH (#ab128915) from Abcam, Cambridge, UK; MCL-1 (#sc-819), HDAC8 (#sc-374180), HSP90 (#sc-13119), vinculin (#sc-73614) from Santa Cruz Biotechnology, Heidelberg, Germany; cleaved caspase-3 (#cs9661), PARP1 (#cs9542), BID (#cs2002), HDAC1 (#cs34589), HDAC2 (#cs5113), histone H3 (#cs14269), ac-histone H3 (K9) (#cs9649), ac-histone H3 (K18) (#cs9675), ac-histone H3 (K27) (#cs8173) from Cell Signaling, Leiden, The Netherlands; ac-tubulin (#T7451) from Sigma-Aldrich, Taufkirchen, Germany; ac-histone H3 (#06-599) from Merck Millipore, Burlington, MA, USA; and NOXA (#ALX-804-408) from Enzo Life Sciences, New York, NY, USA. HSP90, GAPDH, and vinculin served as independent housekeeping proteins to normalize protein loading. The protein ladders used were the PageRuler^TM pre-stained protein ladder (#26616) and the PageRuler^TM Plus pre-stained protein ladder (#26619) from Thermo Fischer Scientific, Waltham, MA, USA.

The organic solvents, inorganic salts, and acids used in the study were manufactured by Dia-m (Moscow, Russia). The sorbent for chromatography Macro-Prep DEAE was purchased from Bio Rad Laboratories, Inc. (Hercules, CA, USA). The phosphate-buffered saline (PBS), L-glutamine, penicillin/streptomycin solution (10,000 U/mL, 10 µg/mL), Minimum Essential Medium Eagle (MEM), and reference standards (mannose, rhamnose, glucose, galactose, xylose, and dextrans) were purchased from the Sigma-Aldrich company (St. Louis, MO, USA). The MTS reagent 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide was purchased from Promega (Madison, WI, USA). The trypsin, fetal bovine serum (FBS), 2-deoxy-D-glucose (2-DG), and the protein marker PageRuler^TM Plus Prestained Protein Ladder were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
The cell lysis buffer (10×), glycolysis antibody sampler kit #8337, antibodies against Glut 1, and horseradish peroxidase (HRP) conjugated secondary antibody from rabbit and mouse were purchased from Cell Signaling Technology (Danvers, MA, USA), and β-actin was purchased from the Sigma-Aldrich company (St. Louis, MO, USA).

For sample analysis, 5 µL of PageRuler^TM Plus Prestained Protein Ladder (Thermo Fisher Scientific, Vilnius, Lithuania) and 500 ng of low and highly phosphorylated Tau proteins were loaded for each condition on a 4%–20% polyacrylamide gel (Mini-PROTEAN^® TGX™, Bio-Rad, Hercules, CA, USA), and allowed to migrate during 1 h at 50 mA in 1× Tris-Glycine-SDS buffer (Bio-Rad). Boiling and β-mercaptoethanol were avoided to preserve multimeric complexes. The transfer was performed at 4 °C for 1 h 45 min and 260 mA (1× Tris-Glycine buffer (Bio-Rad) with 20% methanol) on a 0.45 µm nitrocellulose membrane (Amersham^TM Proton^TM, GE Healthcare, Berlin, Germany) which was incubated overnight with the corresponding primary antibodies diluted in Tris buffered saline (TBS)-Tween-20 0.05% (Sigma-Aldrich, St Louis, MO, USA) containing 5% bovine serum albumin (BSA): rabbit anti-phosphoT231 antibody 1:1000 (Abcam, Cambridge, UK), mouse anti-Tau antibody 1:1000 (Invitrogen, Carlsbad, CA, USA) and mouse anti-His-tag antibody 1:2000 (Invitrogen). Incubation with secondary antibodies was carried out for 1 h, either with an anti-mouse or an anti-rabbit (1:10,000) from sheep coupled to horseradish peroxidase (HRP) (GE Healthcare Biosciences, Uppsala, Sweden) and diluted in TBS-Tween-20 0.05%. All washing steps were accomplished in TBS-Tween-20 0.05%.