Protein assay kit

PDAC cells were treated with different concentrations of raloxifene or solvent in serum-free medium. Phosphorylation was stimulated by IL-6 two hours after the addition of raloxifene. After an incubation period of three, six, or 24 hours, cells were washed with ice-cold phosphate buffer saline (PBS) and lysed in radioimmunoprecipitation assay lysis buffer containing phosphatase and protease inhibitors. Protein concentrations were determined using a Quantipro™ BCA (bicinchoninic acid) protein assay kit (Sigma-Aldrich, Taufkirchen, Germany) according to the manufacturer’s instructions. Next, the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro-transferred to polyvinylidene difluoride membranes (Perkin Elmer, Boston, USA). The resulting membranes were blocked (Tris-buffered saline with Tween-80, 5% bovine serum albumin and 0,02% sodium azide) and incubated with specific primary antibodies overnight at 4 °C and, subsequently, probed with horseradish peroxidase-conjugated secondary antibodies. Signals were visualized by luminescence imaging (Peqlab Biotechnologie GmbH, Erlangen, Germany) using an enhanced chemiluminescent substrate system (SuperSignal™ West Pico Plus, Thermo Fisher, Ulm, Germany).

The plasma and mucosal lysozyme activity was measured using lyophilized Micrococcus luteus according to Ellis [44 ]. The total immunoglobulin (Ig) concentration in plasma and mucus was assayed by polyethylene glycol method [45 ]. The plasma alternative complement activity (C3) was determined by measuring the haemolysis rate of rabbit red blood cells [46 (link)]. The blood bactericidal activity was determined against Streptococcus iniae (OD: 0.5 at 546 nm) inside a bacterial suspension and following calculating bacterial colony forming unites (CFUs) on nutrient agar plates after 24 h incubation at 35°C [47 (link)].
The total protein was determined according to Bradford [48 (link)] using a Sigma-Aldrich Protein Assay Kit. Protease activity in mucus was assayed by the Azocasein hydrolysis procedure, as described by Ross et al. [49 (link)]. Alkaline phosphatase (ALP) activity in mucus was assayed colorimetrically at 405 nm by assay kit (Sigma-Aldrich, CO, USA) based on the hydrolysis of p-nitrophenol phosphate to p-nitrophenol [49 (link)].

Homogenization of the cells was performed on ice 1:10 (w/v) in a buffer containing 50 mM Tris (pH 7.4), 0.1 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin and 10 mg/mL leupeptin. The measurement of protein concentration for each sample was performed according to the Lowry procedure using a protein assay kit (Sigma, St. Louis, MO, USA), and western blotting was performed. Separated proteins with SDS polyacrylamide gel electrophoresis were transferred to nitrocellulose membranes (Santa Cruz Biotechnology, Inc., Texas, and USA). Nitrocellulose blots were blocked with 5% dry milk and probed with Caspase 3 (sc-7272, Santa Cruse Biotechnology, Texas, USA) and Lc-3 (sc-398822, Santa cruse) primary antibodies at a dilution of 1:500. The blots were washed and incubated for 1 h with a secondary antibody, antimouse or antirabbit Ig peroxidase-conjugated (Santa Cruz Biotechnology, Inc., Texas, and the USA) at a dilution of 1:500. Specific binding was detected with chemi-glow detection reagents using chemiluminescent detection. The relative amount of immunoreactive bands on Western blots was quantified in arbitrary units by scanning blots using a computerized software program (LabWorks 4.0; UVP, Inc., Cambridge, UK). Primary human anti-Beta actin and secondary antimouse antibodies were used to monitor Beta-actin expression as a loading control.

Total cells were harvested after treatment, washed twice with cold PBS, and lysed with RIPA lysis buffer (KeyGEN BioTECH, Nanjing, China) including 1% cocktail (Selleck Chemicals, Houston, TX, USA). Lysates were centrifuged at 12,000× g at 4 °C for 15 min, and the supernatant was collected. Total protein concentration was quantified using the bicinchoninic acid (BCA; Beyotime Institute of Biotechnology, Nanjing, China) protein assay kit (Sigma Chemical Co., St. Louis, MO, USA). The proteins were separated electrophoretically using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then the gel was transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were subsequently blocked with the use of 5% milk in tris-buffered saline Tween (TBST) for 1 h and incubated overnight with antibodies against p217/p221-MEK (Pmek, Cell Signaling Technology, Beverly, MA, USA), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Inc., Santa cruz, CA, USA) in TBST containing 5% defatted milk at 4 °C followed by incubation with horseradish peroxidase-linked secondary antibodies (Beyotime Institute of Biotechnology, Nanjing, China) at 4 °C for additional 1 h. The immunobands were detected with an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology, Nanjing, China).

At the end of the experiment, the right main bronchus was subsequently ligated and a catheter was inserted from the trachea into the left lung. Then, the left lungs were lavaged with 1 mL of autodaved PBS for five times .The recovery ratio of the fluid was about 80% (4 mL) and the BALF was immediately centrifuged at 300 g for 10 min at 4 °C and the supernatants were stored in −80 °C for analysis. The cell pellets were re-suspended in PBS, and the total cell number was counted using a standard hemocytometer. Meanwhile, the cell pellets were re-suspended in PBS, centrifuged onto slides, and stained for 10 min with Wright-Giemsa staining. The slides were quantified for macrophages, neutrophils, and lymphocytes by counting a total of 200 cells/slide at ×40 magnification as the differential cell count. The levels of TNF-α in BALF were determined by enzyme-linked immunasorbent (ELISA) kits. The protein concentration in BALF supernatants was measured by the Bradford method by using a Bio-Rad protein assay kit with bovine serum albumin (Sigma) as a standard.

PC12 cells were lysed in a buffer (100 mM Tris-HCl, pH 7.5, 5 M NaCl, 0.5 m EDTA, 10% SDS, 1% NP40, IGEPAL), containing protease and phosphatase inhibitor, and centrifuged at 15,000g for 20 min at 4°C. The supernatant was collected, and protein concentration was determined using a protein assay kit (Sigma). Samples containing 40 μg of total proteins were solubilized and electrophoresed on a 12% sodium dodecyl sulphate- (SDS-) polyacrylamide gel. Following electrophoresis, proteins were transferred to the nitrocellulose membrane (Bio-Rad Laboratories, MI, Italy). The membrane was immersed in a blocking solution (3% nonfat dried milk in 20 mM Tris and 137 mM NaCl at pH 7.6 containing 0.05% Tween-20) for 2 h on a plate shaker. Subsequently, the membrane was incubated with mouse anti-pS6 primary antibody (1 : 2000; Millipore, Burlington, MA, USA) overnight at 4°C on the plate shaker. Blot was probed with horseradish peroxidase-labeled secondary antibodies, and the bands were visualized with enhanced chemiluminescence reagents (Bio-Rad Laboratories). Image analysis was carried out by ChemiDoc System (Bio-Rad Laboratories).