Enhanced chemiluminescence detection system

Protein concentration was determined using the Bradford method (Thermo Fisher Scientific, Waltham, MA, USA). For the examination of protein expression levels in lysed cells, the proteins were separated by SDS-PAGE using 10% acrylamide gel. Then, the separated proteins were transferred onto a nitrocellulose membrane and blocked with 5% (w/v) skim milk in 1 × TBS containing 0.3% (v/v) Tween (TBS-T) at room temperature for 1 h. Membranes were washed twice with TBS-T and incubated with the primary antibody, mouse anti-cyclin D1 (Merck Millipore, Danvers, MA, USA) at 4°C overnight. After being washed five times with TBS-T, membranes were incubated with the horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare Biosciences, Buckinghamshire, NA, UK) at room temperature for 2 h and then washed with TBS-T five times. Immunoreactive material was then visualized with an enhanced chemiluminescence detection system (GE Healthcare Biosciences). To confirm equal protein loading, each membrane was stripped and incubated with mouse anti-β-actin (Sigma-Aldrich).

Three hundred thousand cells of RAW264.7 were cultured in complete RPMI 1640 medium at 37°C. The cells were infected with 1.5 × 10⁷ cells of L. donovani and incubated for 24h at 37°C. After washing three times with PBS, the macrophages were lysed in RIPA Buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma Aldrich). Cell lysates were diluted with SDS sample buffer, boiled for 5 min and separated by electrophoresis on an SDS-containing 4–20% Tris-HCl gradient gel (Thermo), then transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Sciences, USA). After blocking with Block Ace (Sumitomo Dainippon Pharma, Japan), the membrane was probed with anti-SIRPα antibody (RayBiotech, USA; 1:2,000 dilution) and anti-GAPDH antibody (GeneTex, USA; 1:2,500 dilution) diluted with PBS containing 0.05% Tween 20 (PBS-T) plus 10% Block Ace. After washing the membrane with PBS-T three times, it was probed with horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG antibody (GE Healthcare) at 1:5,000 dilution with PBS-T containing 10% Block Ace. Bands were visualized by an enhanced chemiluminescence detection system (GE Healthcare) and analyzed by LAS-3000 mini (Fujifilm, Japan). Densitometric analysis was performed using Image J software from the National Institute of Health.

Whole cell extracts from cell lines were prepared using radio-immunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. Total cell protein (15–20 μg) was used for Western blotting. Samples were transferred to PVDF membranes that were immersed in 3% skim milk containing antibody against CYP1A1 (polyclonal #ab3568, Abcam, Cambridge, MA), AKT (monoclonal #4691, Cell Signaling Technology, Danvers, MA), phospho-AKT (monoclonal #4060, Cell Signaling Technology), BCL2 (polyclonal sc-492, Santa Cruz Biotechnology, Dallas, Texas), and GAPDH (monoclonal sc-47724, Santa Cruz Biotechnology) overnight at 4°C. Blots were washed in TBS containing 0.1% Tween20 and labeled with horseradish peroxidase conjugated, secondary anti-rabbit antibody (Cell Signaling Technology). Specific complexes were visualized with an enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, UK) using the Chemidoc imaging system (Bio-Rad, Hercules, CA). GAPDH was used as control for equal protein loading.

Total protein lysates were obtained using RIPA buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 50 mM Tris (pH 7.6), and 10 μL/mL protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Equal amounts of protein from each sample were separated on SDS-PAGE gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in TBS-T (150 mM NaCl, 10 mM Tris, pH 7.4, and 0.1% Tween-20), and incubated with the following primary antibodies at 4°C overnight: poly [ADP-ribose] polymerase (PARP), cleaved PARP, CHK1, phosphorylated CHK1 (Ser345), pH2A.X, and Rad51 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-CDK 4, anti-cyclin A, -B1, -D1, -E, p21^CIP1, and cdc25A (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), active caspase 3 (1:500; Epitomics, Burlingame, CA), and anti-actin (1:3000; Millipore, Billerica, MA, USA) as a loading control. After several washes with TBS-T, the membranes were incubated with HRP-conjugated secondary antibody (1:5000; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence detection system (GE Healthcare, Wauwatosa, WI, USA).