Uvp biospectrum imaging system

Treated cells were washed twice with PBS, then lysed in 200 μl of RIPA lysis buffer (Millipore, 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor (Roche). Thirty micrograms of protein from the supernatant was loaded on an SDS-polyacrylamide gel, followed by western blot analysis (antibody information is listed in Additional file 1: Table S2). The immuno-reactive bands were revealed by an ECL system (Millipore) then developed and quantified on a UVP BioSpectrum Imaging System. Each band was quantified using ImageJ. β-Actin used as internal (loading) control for relative quantitation.

Cell pellets were re-suspended and incubated in lysis buffer (50 mM Tris 7.5, 0.4 M NaCl, 1% Tween 20, 1% NP40, 50 mM sodium orthovanadate, 0.25 M β–glycerophosphate, 0.5 mM dithiothreitol and protease inhibitors) for 30 min and then sonicated at 4°C. The total protein amount was measured by the Bradford method with a protein assay kit (Bio-Rad, CA, USA). Equal amounts of protein from the lysate were loaded and separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, CA, USA). The blots were hybridized with primary antibodies against caspase 3 (Millipore), β-actin (Cell Signaling Technology, MA, USA), XPC (Cell Signaling Technology), DDB2 (Cell Signaling Technology), p-Rad18 (Cell Signaling Technology), p-ATM (S1981) (Cell Signaling Technology), p-Chk2 (T68) (Cell Signaling Technology), p-DNA-PKcs (S2056) (Cell Signaling Technology), p-Akt (S473) (Cell Signaling Technology), and Akt (Cell Signaling Technology), respectively. After incubation with the horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), the horseradish peroxidase activity was detected by an enhanced chemiluminescence system (UVP BioSpectrum Imaging System).

The mice were kept in individual cages with filter paper lining the bottom (Solarbio, China) for 3 hours. During the test, the mice were fed with dry mouse chow, but water was withheld. The number of urinary spots was examined and recorded using an ultraviolet light camera (UVP BioSpectrum Imaging System, USA). ImageJ software (NIH, USA) was used to analyze the spots. Urinary volume is linearly associated with the area of the spot (R=0.9989). Sporadic noncircular small-diameter urine spots (particle<0.01475 cm², corresponding to 0.5 μL urine) were excluded.

For Western blotting, total cellular proteins were prepared from the thyroid tissues by the method described previously [27 (link)]. Fifty micrograms of sample protein was separated with 12% SDS/PAGE, and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in NaCl/Tris-T (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 0.5% Tween-20) at 4 °C overnight. It was rinsed three times (10 min each) with NaCl/Tris-T, and this was followed by 3 h of incubation with the same first antibodies that were used in immunohistochemical staining in the appropriate concentrations (IL-6, 1:500; COX-2, 1:500; NF-κB/p65, 1:500; β-actin, 1:3000, and IkBα, 1:1000) and 1 h of incubation with horseradish peroxidase-conjugated anti-rat IgG (Zymed Laboratories, San Francisco, CA, USA). Immunolabeling was detected with an enhanced chemiluminescence system (Roche, Mannheim, Germany), and visualized with the UVP Bio-spectrum Imaging System (UVP, Upland, CA, USA). β-actin was used as the internal quantitative control in densitometry analyses.

Cells were lysed with lysis buffer containing complete protease inhibitor cocktail (Roche Life Science, Indianapolis, IN, USA). Cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with the indicated primary antibody and then with the HRP-conjugated secondary antibody. Visualization of the immunoreactive bands was performed using chemiluminescent HRP substrate (Millipore, Temecula, CA, USA) and UVP BioSpectrum Imaging System.

After protein transfer, the nitrocellulose membranes were blocked in 1% bovine serum albumin (BSA), incubated with the appropriate primary antibodies and developed using enhanced chemiluminescence. The membranes were probed with the following primary antibodies: Mkrn3 (ab177203), obtained from Abcam (Abcam, Cambridge, MA) and β-Actin (monoclonal AC-15, A1978) from Sigma (Sigma, St. Louis, MO, USA). HRP-conjugated IgG Goat Anti-mouse and Goat Anti-rabbit (Jackson ImmunoResearch, PA USA) secondary antibodies were used. The anti-MKRN3 antibody was used in 1:1,000 dilution and the anti-ACTIN antibody was used in 1:10,000 dilution in a solution of 1% non-fat dried milk in TBS/Tween. Secondary antibodies were used in a 1:20,000 dilution in non-fat dried milk in TBS/Tween. The levels of Mkrn3 protein were normalized with the corresponding β-Actin loading control within the same immunoblot. Visualization of the immunoblots and acquisition of the images was carried out the UVP BioSpectrum Imaging System (UVP, LLC, Upland, CA, USA). Dynamic Total Time Exposure settings were used, with binning set at 12.1 MP, 149% and 1 × 1 interpolation. Exposure times varied depending on the primary antibody used.

For Western blotting, total cellular proteins were prepared from the cells by the method described previously [23 (link)]. Fifty micrograms of sample protein was separated with 12% SDS/PAGE and transferred to a polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in NaCl/Tris-T (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, and 0.5% Tween-20) at 4°C overnight. It was rinsed three times (10 min each) with NaCl/Tris-T, and this was followed by 2 h of incubation with the first antibodies in the appropriate concentrations (β-actin, 1 : 3000; SOD2, 1 : 4000; Cat, 1 : 3000; pro-caspase-9, 1 : 300; pro-caspase-3, 1 : 500; active-caspase-9, 1 : 300; active-caspase-3, 1 : 500; SULT1A1, 1: 1000; and SULT1C2, 1: 600) and 1 h of incubation with horseradish peroxidase-conjugated anti-rat IgG (Zymed Laboratories, San Francisco, CA, USA). Immunolabeling was detected with an enhanced chemiluminescence system (Roche, Mannheim, Germany) and visualized with the UVP BioSpectrum Imaging System (UVP, Upland, CA, USA). ImageJ was used to measure the density of bands (National Institutes of Health, Bethesda, MD). β-Actin was used as the internal quantitative control in densitometry analyses.