Enhanced chemiluminescent reagent

Cell lysates were prepared using NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris, pH 8) with protease inhibitor cocktail (Hoffmann La Roche Ltd, Basel, Switzerland). Protein concentration was measured using the Bradford protein assay and 20 μg of total protein was loaded in each well. SDS-PAGE gel electrophoresis was used to separate the proteins. Using wet transfer, proteins were transferred onto a nitrocellulose membrane. After transfer, the membranes were probed with mouse antibodies against smooth muscle actin, vimentin (Dako), c-MYC (Abcam Ltd, Cambridge, UK), and actin (Sigma-Aldrich), and rabbit antibodies against OCT4 (Novus Biologicals, Littleton, CO, USA), SOX2 (Abcam Ltd), and E-cadherin (Cell Signaling Technology). Secondary antibodies (horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG) were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Immunocomplexes were visualized using enhanced chemiluminescent reagents (GE Healthcare Bio-Sciences AB).

U87 and U373 glioma cell lysates were prepared in 0.1% Triton buffer containing protease and phosphatase inhibitors (Sigma-Alrich) and also were prepared cytoplasmic and nuclear extracts using cell compartment isolation kit (Millipore). Further total protein was quantitated using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Blots were exposed to anti-GSK3 (Millipore), anti-phosphotyrosine GSK3 (Millipore), anti-phospho-ERK1/2 (Thr202/Tyr204) (Millipore), anti-α-tubulin, and anti-beta-catenin (Millipore) and recognized by an HRP-conjugated horse anti-mouse secondary antibody (Santa Cruz Inc); antibodies to detect phospho-AKT1/2/3 (Ser473), total Akt1, hnRNPA1, and Anxa7 (Santa cruz Inc) SF2/ASF1 (Santa Cruz Inc) were recognized by an HRP-conjugated goat anti-rabbit secondary antibody (Santa Cruz Inc.). Enhanced chemiluminescent reagent (GE Healthcare) was added to the membranes according to the manufacturer's protocol and visualized by autoradiography.

Total protein in each sample was measured by the BCA assay (Bio-Rad, USA) using bovine serum albumin as the standard protein. A fixed amount of protein (20 μg) from each sample was fractionated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Pall, USA). Membranes were incubated in 5% milk in TBS for 1 h in room temperature. For identification of β-ARs, membranes were exposed to primary antibodies (anti-β1-AR, 1 : 250 dilution, or anti-β2-AR, 1 : 500 dilution, abcam, USA). After washing, the membranes were incubated with secondary antibodies (HRP-conjugated anti-rabbit antibody, 1 : 5000 dilution, KBL, USA). The bound antibodies were detected using the enhanced chemiluminescent reagent (GE Health Care, USA). Data are presented as the β-ARs to actin ratio and then expressed as fold-change compared to WKY group.

Cells were lysed in 30 µl of protein lysis buffer (50 mM Tris, 10% v/v glycerol, 50 mM NaF, 1 mM EGTA, 1 mM EDTA, 10 mM glycerol phosphate, 1% v/v Triton X-100, 1× complete inhibitor cocktail, 1 µM microcystin-LR and 1 mM Na₃VO₄). Ten µg of protein were electrophoresed through a 12% bis-acrylamide gel and transferred to a polyvinylidene fluoride membrane (GE Healthcare). The membrane was then blocked and probed with antibodies against BMPR-II (goat polyclonal; Santacruz, cat no. sc-5682), BMPR-IA (rabbit polyclonal; Santacruz, cat no. sc-20736), BMPR-IB (rabbit polyclonal; Santacruz, cat no. sc-25455) or phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428; rabbit polyclonal; Cell Signalling, cat no. 9511), overnight. Anti-β-actin (Sigma, cat no. A5316) antibody was used as a loading control. Enhanced chemiluminescent reagent (GE Healthcare) was used for visualisation of the proteins using a G:BOX gel doc system (Syngene).

Cells were harvested in cell lysis buffer, and proteins were separated by 10% SDS-PAGE. The target proteins were detected using enhanced chemiluminescent reagent (GE healthcare, USA). The antibodies used for immunoblotting were anti-BAG5 (Santa Cruz, USA), anti-p53 (Santa Cruz, USA), anti-HIF-1α (BD Biosciences, USA), anti-α-synuclein (Genetex, USA), anti-HSP70 (Santa Cruz, USA), anti-phospho-p53 (Ser15) (Cell Signaling, USA), anti-phospho-α-synuclein (Ser129) (Abcam, UK), and anti-β-actin (Sigma-Aldrich, USA). The autoradiographic films of Western blots were scanned by a Microtek ScanMarker i900, and the protein bands were quantified by ImageJ software. To convert the amounts of BAG5 from etoposide and H₂O₂ stimulation to the equivalent BAG5 expression from plasmid transfection, Western blots were quantified as above. Linear regression was calculated using the ImageJ scores from different amounts of BAG5 plasmid transfection after normalization with the β-actin scores. The amounts of etoposide- and H₂O₂-induced BAG5 were then calculated by the linear regression equation [30 (link)].

Lymphocyte separation media (LSM), Lymphosep, was obtained from BioWest S.A.S (Nuaillé, France). Protease inhibitor (Complete, Ultra, Mini, EDTA-free) and phosphatase inhibitor (PhosStop) cocktails were obtained from Roche (Mannheim, Germany). Luminol and p-coumaric acid were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). The commercially available kit for determination of total cholesterol Beckman enzymatic reagent kit was purchased from Beckman Coulter (Brea, CA, USA). The nitrate/nitrite colourimetric assay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). For measuring the activity of arginase, we used L-arginine monohydrochloride obtained from Kemika (Zagreb, Croatia) and alpha-isonitrosopropiophenone (ISPF) obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). The polyclonal rabbit anti-iNOS and anti-NFκB-p65, monoclonal mouse anti-actin antibody, secondary anti-mouse, and anti-rabbit IgG horseradish peroxidase-linked antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The following antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, US): anti-phospho-NFκB-p65 (Ser⁵³⁶), anti-phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), anti-total ERK1/2, anti-phospho-Akt (Ser⁴⁷³), and anti-total Akt antibodies. The enhanced chemiluminescent reagent was obtained from Amersham, GE Healthcare (Buckinghamshire, UK).