Las500

Western blot analyses were performed as described previously (23 (link)). To detect band shifts that represent phosphorylated proteins, SDS-PAGE gels containing 50 μm Phos tag acrylamide and 100 μm manganese ions (Mn²⁺) were used. The Phos tag forms a complex with two Mn²⁺ ions and serves as a phosphate-binding molecule (24 (link)). The complex is used for phosphate affinity SDS-PAGE, which results in the upward mobility shift of phosphorylated proteins. Reacted proteins were detected using the LAS4000 or LAS500 imaging systems (GE Healthcare).

Protein concentrations in samples were measured using the BCA Protein Assay Regent (Micro BCA Protein Assay Reagent kit, 23225, Thermo Fisher Scientific, Waltham, MA, USA). After the membranes (Immobilon-P, IPVH00010, Merck Millipore, Burlington, MA, USA) were washed by TBS (20 mM Tris-HCl/pH 7.5, 500 mM NaCl), samples of 2.5 μg/25 μL were dropped on membranes using Bio-Dot Apparatus (1706545, Bio-Rad Laboratories Laboratories, CA, USA) and left to stand overnight. Next, the membranes were blocked with 5% skim milk in TBST (10 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20), and reacted with the following primary and secondary antibodies diluted in Can Get Signal solution (NKB-101, Toyobo, Osaka, Japan). ECL prime (RPN2232, GE Healthcare, Chicago, IL, USA) or ECL select (RPN2235, GE Healthcare, Chicago, IL, USA) were used to detect the bands using LAS500 (29005063, GE Healthcare, Chicago, IL, USA). Primary and secondary antibodies were diluted as follows: mouse anti-amyloid β, (1:5000, clone 82E1, #10323, IBL, Gunma, Japan); HRP-conjugated anti-mouse IgG (1:5000, NA931VA, GE Healthcare, Chicago, IL, USA).

After treatment, cells were washed twice with ice-cold phosphate-buffered saline (PBS) (Nissui Pharmaceutical, Tokyo, Japan) and harvested in RIPA buffer [25 mM Tris (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate 0.1% SDS; Thermo Fisher Scientific, Waltham, MA, USA]. Protein concentrations were measured using the BCA protein assay kit (Thermo Fisher Scientific). Protein samples were separated on SDS-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. The membrane was probed with primary antibodies and subsequently probed with horseradish peroxidase-conjugating (HRP) secondary antibodies (1:10000; GE Healthcare, Little Chalfont, UK). Proteins were detected by enhanced chemiluminescence using ImmunoStar® Zeta (Wako Pure Chemical Industries). The chemiluminescence images were taken using the LAS-500 or LAS-4000 (GE Healthcare) device. Primary antibodies were purchased as follows: GAPDH (1:2000) from American Research Products (Waltham, MA, USA); Lamin A/C (1:1000), ARNT (1:1000), BIRC3 (1:1000), caspase-3 (1:1000) and cleaved caspase-3 (1:1000) from Cell Signaling Technology (Danvers, MA, USA); and β-actin (1:1000) from Sigma-Aldrich.

Cells were lysed by lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% Triton-X 100, protease inhibitor (Roche)) and disrupted by a sonicator. Cell lysates were centrifuged at 15,000 rpm and soluble supernatants were separated. 15–30 μg of cell lysates were mixed with sample buffer (60 mM Tris, 25% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.05% bromophenol blue) and separated by 10% SDS-PAGE. The proteins were transferred to nitrocellulose membrane. Membranes of each protein size were probed with the primary antibodies and the corresponding secondary antibodies. The signals were detected using LAS-500 (GE Healthcare) according to manufacturer’s protocol. All signals were quantified using Image J software^{54 (link)}. Following antibodies were purchased: anti-AR (Cat# SC-7305, Santa Cruz Biotechnology), anti-AR-V7 specific (Cat# 68492S, Cell signaling), anti-PSA (Cat# ab53774, Abcam), anti-GAPDH (Cat# A300-641A, Bethyl), anti-Lamin A/C (Cat# 2032, Cell signaling).

For fractionation of 30 μg of the promastigote protein extract from each Leishmania species at the logarithmic phase, SDS-PAGE 12% gel was performed. The protein bands were transferred onto nitrocellulose membranes (Hybond, Amershan, UK) in a trans-blot semidry transfer unit (GE Healthcare) by applying a current of 1.6 mA/cm² for 2 h. The membranes were rinsed with PBS–Tween 0.1% and incubated with blocking buffer (5% low-fat milk powder in PBS–Tween 0.1%) at 4°C for 1 h. The transblotted proteins were probed overnight with a rabbit polyclonal anti-arginase antibody. Then, the membrane was washed with PBS–Tween 0.5% for 5 min thrice, and incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies (1:1000 diluted). Specific binding was revealed with a western blotting detection ECL system (Amersham, UK) and exposed to LAS 500 (GE Healthcare, Life Sciences). The band intensities were quantified using the ImageJ 1.41 software (NIH, USA).
The gel loading control was determined by the number of parasites subjected to protein extraction, dosage of the protein extract, and application of the same amount of proteins in each well, and also by the quantification of the SDS-PAGE bands stained by Coomassie Brilliant Blue G250 [31 (link)].