Pierce bca reagent

Corresponding cell lysates was extracted from bladder cancer tissues and cell lines using RIPA buffer (Thermo Fisher Scientific Inc., Rockford, IL USA) containing protease inhibitors cocktail (Roche) according to the manufacturer's protocol. Accurate protein concentration was determinate by Pierce BCA Reagents (Thermo Fisher Scientific Inc.). Western blotting was carried out using the same antibody as for the immunohistochemistry analysis (dilution: 1:1,000). Additional primary antibodies such as rabbit anti-tubulin (dilution: 1:1,000) were used.
Briefly, 30 μg of protein lysates from each sample was separated by electrophoresis in 10% sodium dodecyl sulfate (SDS) polyacrylamide gel before being transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) for 2 hours. Membranes were blocked using 5% bovine serum albumin (BSA), and incubated in TBST (Tris buffered saline with 0.05% tween) containing rabbit polyclonal IgG2a anti-PBRM1 (1:1000, Sigma, molecular weight: 181 kDa) or anti-tubulin (1:1,000; cell signaling technology, molecular weight: 55 kDa) overnight at 4°C. The membranes were then incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin (1:1000, Cell Signaling Technology) as secondary antibody and visualized using a commercial ECL kit (Pierce, Rockford, IL, USA).

Snap-frozen tissues were homogenized using MAGNAlyser beads (Roche Life Science, Penzberg, Germany) in RIPA lysis buffer (Sigma-Aldrich) with protease inhibitors and phosphatase inhibitors (Nacalai Tesque Inc, Kyoto, Japan). The samples were centrifuged for 20 min and the supernatant homogenate collected. Protein concentrations were determined using the Pierce BCA reagent (Thermo Fisher).
Luminex ELISA was carried out for the detection of murine interleukins, chemokines, and growth factors (R&D Systems, MN; LXSAMSM-21). Sample preparation followed the manufacturer’s protocol, and analytes were read using the MAGPIX Platform (Luminex Corporation, TX).
For blotting, 30  μg samples were separated by SDS-PAGE (Bio-Rad Laboratories Inc, CA) and transferred to PVDF membranes (Bio-Rad Laboratories Inc). The PVDF membranes were blocked with 5% blotting grade blocker (Bio-Rad Laboratories Inc) at room temperature (RT) for 1  hr and then incubated with the primary antibodies (anti-myoglobin antibody, 25919S, Cell Signaling Technology) overnight at 4°C. Then, the membranes were washed with TBST at RT and incubated with the secondary antibody for 1  hr. Protein bands were detected with Pierce ECL substrate (Thermo Fisher) according to the manufacturer’s instructions. Data were analyzed with the ChemiDoc Imaging System (Bio-Rad Laboratories Inc).

293T cells were washed with PBS and lysed with PBS containing 1% NP-40. Protein concentration was determined using Pierce BCA reagent (ThermoFisher; 23221, 23224). 35 μg protein was loaded in each lane of 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membrane and immunoblotted with the following primary antibodies: anti-Bax antibody (Santa Cruz N20), anti-Bak antibody (Cell Signaling 3814), anti-Bid antibody (R&D Systems AF860), anti-Bim antibody (Sigma B7929), anti-Puma antibody (Cell Signaling 4976), anti-Bcl-2 antibody (Abcam 32124), anti-Bcl-xL antibody (Cell Signaling 2764), and anti-Mcl-1 (Santa Cruz S19) at 1:1,000 dilution. The secondary anti-rabbit and mouse antibodies, conjugated with HRP, were obtained from Santa Cruz and were used at 1:2,000 dilution. The luminescence signal was detected using ECL reagent (ThermoFisher 32106).

Western blots were carried out as previously described [40 (link),41 (link)]. Briefly, cells were washed once in PBS and collected by scraping into 100 μL of ice-cold lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1 mM PMSF, and protease inhibitors). The extracts were further lysed with sonication and the supernatants were collected after centrifugation. Protein concentrations were determined with the Pierce BCA reagent (Thermo Fisher, Budapest, Hungary). Proteins (30 μg/lane) were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 90 min. Primary antibodies against iNOS (rabbit monoclonal; Sigma-Aldrich, Budapest, Hungary) were applied overnight at 4 °C. After three washes in TBS containing 0.05% Tween 20, the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, Sigma-Aldrich, Budapest, Hungary) was applied for 1 h. Blots were washed in TBS containing 0.05% Tween 20 three times and once in TBS. After washing, the blots were incubated in Pierce Supersignal Chemiluminescent reagent (Thermo Fisher, Budapest, Hungary). For detection of luminescence, Chemidoc Touch gel documentation system (BioRad) was used and signals were quantified by densitometry using the ImageLab 6.0 software.