Image analysis system

For western blot analysis, the kidneys were removed and snap-frozen in liquid nitrogen for protein extraction using RIPA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Next, 20 μg crude protein was subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following electrophoresis, the proteins were transferred onto a nitrocellulose membrane (GE-Healthcare, Little Chalfont, UK). The blots were blocked with 2% bovine serum albumin in phosphate-buffered saline (PBS), which was followed by 1 h incubation with 1:2,000 diluted primary antibodies against phosphorylated-p38 mitogen-activated protein kinase (p-p38 MAPK), phosphorylated-c-Jun N-terminal kinase (p-JNK) or NF-κB p65 (Santa Cruz Biotechnology, Inc.). Following washing with PBS, the reacted blots were incubated with peroxidase-conjugated secondary antibodies (BioSource International, Inc., Camarillo, CA, USA). Antigen-antibody complexes were identified by enhanced chemiluminescence (Pierce Biotechnology, Inc.) and the photographic density was quantified using an image analysis system (Fujifilm, Tokyo, Japan). GAPDH and lamin B1 were used as internal controls.

After treatment, the cells were detached by trypsinization, collected by centrifugation, and then lysed in the PBS lysis buffer containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The cell lysates were centrifuged at 20,000 x g at 4 °C for 10 min, and then the supernatant was collected and used as a crude extract. The protein concentration in the extracts was assessed by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Extracts containing equal protein (30 µg) were separated by electrophoresis on an SDS-polyacrylamide gel. The electrophoresed proteins were transferred to a nitrocellulose membrane (PROTRAN BA85, 0.45 μm; Sigma) with a Transphor Unit (Bio-Rad Laboratories, Hercules, CA, USA). The transferred membrane was blocked with 1% (w/v) BSA in PBS followed by a 1-h incubation with the primary antibodies and then the secondary antibodies. The signal was developed with ECL chemiluminescence reagent (SuperSignal West Dura HRP Detection Kit; Pierce Biotechnology, Rockford, IL, USA), and the signals were acquired and quantitated with an image analysis system (Fujifilm, Tokyo, Japan).

Cells were lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min, centrifuged at 20,000× g at 4 °C for 15 min to remove cell debris, and then the supernatant was used as crude protein extract. A protein assay was conducted using the Bradford method according to the manufacturer’s instruction (Bio-Rad Laboratories, Hercules, CA, USA). The crude proteins were separated using SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Immobilon, Merck, Billerica, MA, USA). The membrane was incubated with 3% (w/v) BSA in PBS for 1 h and then incubated with primary antibodies (1000-fold dilution) for 2 h. Thereafter, the membrane was washed with PBS containing 0.5% Tween-20 (PBST) and incubated with secondary antibodies (2000-fold dilution) for 2 h. The bound antibodies were detected using an ECL chemiluminescence reagent (SuperSignal West Dura HRP Detection Kit; Pierce Biotechnology, Rockford, IL, USA), and the resulting chemiluminescence signals were recorded and semi-quantitated with an image analysis system (Fujifilm, Tokyo, Japan). Signals from DMSO treatment were used as control.

Cells were scraped off into culture medium and collected by centrifugation at 4000
g for 15 min before resuspension in the lysis buffer. Cell lysate
was subjected to a 12% SDS-PAGE gel and subsequently transferred onto a
nitrocellulose membrane as previously described. The resulting blot was incubated in
5% nonfat milk in PBS for 1 h and then probed with a primary antibody against
collagen type I and NF-κB p65, followed by reacting with an appropriate
peroxidase-conjugated secondary antibody for 1 h. Signals were detected using an
enhanced chemiluminescence detection kit (Amersham Biosciences, USA) and were
quantitated by an image analysis system (Fuji Film, Japan).

Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min. After centrifugation (20,000× g, 4 °C, 15 min) to remove insoluble debris, the supernatant (crude protein extracts) was collected for the following analysis. Protein concentration was assessed by Bradford method in accordance with the manufacturer’s instruction (Bio-Rad Laboratories, Hercules, CA, USA). Crude proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (Immobilon, Merck-Millipore, Bedford, MA, USA), and then blocked with 3% (w/v) BSA/PBS at 25 °C for 1 h. Thereafter, the membrane was incubated with primary antibodies (1000-fold dilution) for 2 h and then incubated with secondary antibodies (2000-fold dilution) for 2 h after being washed with 0.5% Tween-20/PBS. The antibody–antigen complex was detected using an ECL chemiluminescence reagent (SuperSignal West Dura HRP Detection Kit; Pierce Biotechnology, Rockford, IL, USA), and the resulting signals were acquired and semi-quantitated using an image analysis system (Fujifilm, Tokyo, Japan). Signals from PBS treatment were used as control.

Cells were lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min. The resulting lysates were centrifuged at 20,000× g at 4 °C for 10 min for the removal of insoluble debris, and then the supernatant was used for the protein assay using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA), and for electrophoresis using an SDS-polyacrylamide gel. The electrophoresed proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) and the membrane was blocked with 2% (w/v) skimmed milk in PBS. The blocked membrane was incubated with primary antibodies (1000-fold dilution) for 2 h, washed with PBS containing 0.5% Tween-20, and incubated with secondary antibodies (2000-fold dilution) for 2 h. The bound antibodies were detected using an ECL chemiluminescence reagent (SuperSignal West Dura HRP Detection Kit; Pierce Biotechnology, Rockford, IL, USA), and chemiluminescence signals were acquired and semi-quantitated with an image analysis system (Fujifilm, Tokyo, Japan). The results of the semi-quantitative analysis are presented as percentage of control (DMSO treatment).