High metastatic NPC 5-8F and non-metastatic NPC 6-10B cell lines were cultured in RPMI-1640 medium (GibcoLife Technologies) supplemented with 10% festal bovine serum(FBS, GibcoLife Technologies) in a humidified chamber with 5% CO2 at a temperature of 37°C. On the one hand, cells were lysed in RIPA lysis buffer, containing 7 M urea, 2 M thiourea, 65 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride (ComWin Biotech) to extract cell extracts. On the other hand, after growing to approximately 70% confluence in 250-mm culture dishes (NEST Biotechnology), cells were washed with 10 ml serum-free medium (RPMI-1640 medium) for three times, and incubated in serum-free medium at 37 °C for 24 h. After incubation, the conditioned medium was collected and centrifuged at 900 × g for 10 min to eliminate suspended cells. The supernatants were concentrated and desalted using Amicon Ultra-15 tubes (molecular mass cutoff, 3KDa; Millipore), followed by addition of proteinase inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride (PMSF), Auragene Bioscience). The concentration of the proteins in cell extracts and supernatants were determined using the BCA protein assay reagent (Tiangen biotechnology). The proteins in cell extracts and collected conditioned media were then stored at −80 °C until use.
Ripa lysis buffer
Manufactured by CoWin Biotech
Sourced in China
RIPA lysis buffer is a commonly used detergent-based buffer solution designed for the extraction and solubilization of proteins from cells and tissues. It aids in the disruption of cell membranes and the release of cellular contents, including proteins, for subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ecl assay kit, by Beyotime (4 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 15 tube, by Merck Group (1 mentions)
Bca protein assay reagent, by Tiangen Biotech (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by CoWin Biotech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Lipo3000, by Thermo Fisher Scientific (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
Bca assay, by Dingguo (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti bax, by Beyotime (1 mentions)
15 protocols using ripa lysis buffer
1
Metastatic and Non-metastatic NPC Cell Line Cultivation and Protein Extraction
2
Western Blot Analysis of Protein Samples
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The treated cells were washed twice with cold PBS and lysed with RIPA lysis buffer (ComWin Biotech, Beijing, China) containing 1% phosphatase inhibitor (ComWin Biotech) and 1% protease inhibitor (ComWin Biotech). The total protein concentration was determined by the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (30 µg per load) of protein samples were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The blots were blocked in 5% non-fat milk for 1 h, and the membranes were washed and incubated with the primary antibody overnight at 4°C, after shaking. After washing with TBS-T, the membranes were incubated with secondary antibody at room temperature for 1 h. The proteins were visualized using an ECL Kit (EMD Millipore), and signal detection was performed in a Bio-Rad Universal Hood 2 Electrophoresis Imaging Cabinet.
3
Quantitative Western Blot Analysis of CTPG-Treated HCC
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Western blot was done according to our previous study.17 (link) HCC were treated with CTPG for 24 hours. After washing with ice-cold PBS
twice, all adherent and floating cells were collected and lysed in RIPA Lysis
Buffer (Beijing ComWin Biotech Co., Ltd) for 20 minutes on ice. After
centrifugation at 12 000 rpm 4°C for 10 minutes, protein concentration was
determined using a Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, USA)
according to the manufacturer’s instructions. The same concentration of proteins
was separated by 12% SDS-PAGE and transferred to PVDF membranes. After washing
with PBST buffer (PBS with 0.05% Tween-20), the membranes were blocked with 5%
non-fat milk at 37°C for 1 hour, and then incubated with the primary antibodies
(Cell Signaling Technology, MA, USA) at proper dilutions overnight at 4°C. After
washing 3 times with PBST, the membrane was incubated with the corresponding
HRP-conjugated secondary antibodies (eBioscience) for 2 hours at 37°C. The
target proteins were detected using ECL assay kit (Beyotime). Grayscale scanning
data were obtained by Image J.
twice, all adherent and floating cells were collected and lysed in RIPA Lysis
Buffer (Beijing ComWin Biotech Co., Ltd) for 20 minutes on ice. After
centrifugation at 12 000 rpm 4°C for 10 minutes, protein concentration was
determined using a Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, USA)
according to the manufacturer’s instructions. The same concentration of proteins
was separated by 12% SDS-PAGE and transferred to PVDF membranes. After washing
with PBST buffer (PBS with 0.05% Tween-20), the membranes were blocked with 5%
non-fat milk at 37°C for 1 hour, and then incubated with the primary antibodies
(Cell Signaling Technology, MA, USA) at proper dilutions overnight at 4°C. After
washing 3 times with PBST, the membrane was incubated with the corresponding
HRP-conjugated secondary antibodies (eBioscience) for 2 hours at 37°C. The
target proteins were detected using ECL assay kit (Beyotime). Grayscale scanning
data were obtained by Image J.
4
Transfection of 293T Cells with Lipo3000
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The 293T cells were cultured in 6 well plates and transfected when the cell density was 60%-70%. Before the transfection, the culture medium was replaced with fresh one. The transfection system was included A (2 μg plasmid, 125 μl Opti-MEM) and B [3.75 μl Lipo3000, 4 μl P3000, 125 μl Opti-MEM (Thermo Fisher, USA)]. The A and B tubes were evenly mixed and kept at room temperature for 15 min. The mixture was slowly added to 6 well plates and mixed. The culture medium was replaced after 8 h. After 36 h, cells were collected and total protein was isolated by the RIPA Lysis Buffer (Beijing ComWin Biotech Co., Ltd). The expression of related proteins was detected by Western blot. The expression of EGFP proteins was also observed by inverted fluorescence microscope (Nikon, Japan).
5
Renal Protein Expression Analysis
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Total protein lysates from renal cortex and HK-2 cells were prepared using RIPA lysis buffer (ComWin Biotech, Beijing, China) with 1% protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay (Dingguo, Beijing, China) according to the manufacturer's protocol. Lysates containing 20–50 μg of protein were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SolarBio, Beijing, China) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBS-T before incubation with primary antibodies specific for β-actin (1:1,000) used as the control, STAT1 (1:1,000), TGF-β1 (1:1,000), Col IV (1:500), and FN (1:500) overnight at 4°C with gentle shaking. The membranes were washed 3 times (10 min each time) with TBS-T, and incubated with goat anti-rabbit secondary antibody (1:10,000) at room temperature for 1 h. Densitometry was carried out using Image J.
6
Western Blot Analysis of Protein Expression
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Total protein lysates were extracted from the mouse renal cortex tissue or cultured podocytes using RIPA lysis buffer (ComWin Biotech, China). Protein samples were sonicated five times for 1 s each, centrifuged at 12 000 rpm for 10 min at 4°C, and then boiled for 5 min. Protein samples were separated by 10-12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (General Electric Co). After being blocked with 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 for 1 h, the membranes were incubated with primary antibody at 4°C overnight and then incubated with secondary antibody at room temperature for 1 h. Details regarding primary and secondary antibodies are listed in Table 2 . The blotted proteins were quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). β-Actin was set as an internal control. The relative expression level of each target protein was displayed as a ratio of target protein/β-actin protein. All the assays were performed at least in triplicate independently.
7
Quantification of Apoptosis-related Proteins
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Anti-caspase-3, anti-cleaved caspase-3, anti-caspase-8, anti-cleaved-caspase-8, anti-caspase-9, anti-cleaved-caspase-9, anti-PARP, anti-cleaved PARP, anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology. Anti-BCL-2 and anti-BAX were obtained from Beyotime Biotech Co., Ltd (Shanghai, China). Anti-β-actin was purchased from Beijing ComWin Biotech Co., Ltd (Beijing, China).
B16-F10 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) and lysed in RIPA Lysis Buffer (Beijing ComWin Biotech Co., Ltd) for 20 min on ice. After centrifugation at 14000g for 10 min at 4 °C, the supernatants were collected and protein concentration was determined by BCA Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Samples were isolated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with PBST buffer (Phosphate Buffered Saline and 0.05% Tween-20) contained 5% nonfat milk for 1 h at RT, and then incubated with corresponding primary antibodies on shaker overnight at 4 °C. After washing three times with PBST, the membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies against mouse or rabbit for 1 h at RT. After extensive washing with PBST, the target proteins were detected using ECL assay kit (Beyotime, China).
B16-F10 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) and lysed in RIPA Lysis Buffer (Beijing ComWin Biotech Co., Ltd) for 20 min on ice. After centrifugation at 14000g for 10 min at 4 °C, the supernatants were collected and protein concentration was determined by BCA Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Samples were isolated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with PBST buffer (Phosphate Buffered Saline and 0.05% Tween-20) contained 5% nonfat milk for 1 h at RT, and then incubated with corresponding primary antibodies on shaker overnight at 4 °C. After washing three times with PBST, the membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies against mouse or rabbit for 1 h at RT. After extensive washing with PBST, the target proteins were detected using ECL assay kit (Beyotime, China).
8
Protein Extraction and Western Blot Analysis
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The collected cell samples were lysed in RIPA lysis buffer (ComWin Biotech Co, Beijing, China) prepared with protease inhibitors (Roche, Basel, Switzerland), and then were placed on ice for 30 min. The lysed cells were centrifuged at 12,000×g for 20 min at 4 °C, and the supernatant were collected. Protein concentrations were determined by the BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). The supernatant (20 μg/well) was mixed with 5 × SDS-PAGE loading buffer and boiled in a 100 °C water bath for 5–10 min. The protein samples were subjected to gel electrophoresis and then transferred to PVDF membranes. After blocking with 10% skimmed milk powder for 2 h at room temperature, the membranes were incubated with anti-parkin antibody (1:1000, Millipore, Massachusetts, USA), anti-DMT1 antibody (1:800, OriGene, Rockville, MD, USA), anti-β-actin antibody (1:10,000, Bioss, Woburn, MA, USA), anti-HA antibody (1:2000, Beyotime Biotechnology, Shanghai, China), anti-Myc antibody (1:2000, Beyotime Biotechnology, Shanghai, China) and anti-Flag antibody (1:2000, Beyotime Biotechnology, Shanghai, China) overnight at 4 °C. Membranes were incubated with a corresponding secondary antibody (1:10,000, Bioss, Beijing, China) for 1 h at room temperature. Finally, blots were imaged using a BioSpectrum Imaging System (UVP, Upland, CA, USA) and quantified using ImageJ software.
9
Molecular Signaling Pathways in UNBS5162 Treatment
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The cells were treated with UNBS5162 for 24 hours and then collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beijing ComWin Biotech Co. Ltd. [CWBIO], Beijing, China). The protein concentration was detected using a BCA kit (CWBIO). Subsequently, the equal proteins (20 μg) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (Bio‐Rad, Hercules, CA, USA) and then transferred onto a polyvinylidene difluoride membrane.17 After blocking with 5% non‐fat dry milk for one hour, the proteins were incubated with various antibodies overnight at 4°C, including Bcl‐2, Bax, Bim, active‐caspase3, AKT, p‐AKT, mTOR, p‐mTOR, and GAPDH (Proteintech Group Inc., Rosemont, IL, USA). Secondary anti‐mouse or anti‐goat horseradish peroxidase antibodies (1/2000 dilution) were added for one hour, and then the bands were visualized using electrochemiluminescence reagents and developed using Hyperfilm‐ECL (GE Healthcare, Piscataway, NJ, USA).
10
Western Blot Analysis of Renal Proteins
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Total protein lysates were extracted from rat renal cortex tissue or cultured podocytes using RIPA lysis buffer (ComWin Biotech). Protein samples were sonicated five times for 1 s each, centrifuged at 12000 rpm for 10 min at 4°C, and then boiled for 5 min. Protein samples were separated by 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (General Electric Co.). After being blocked with 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 for 1 h, the membranes were incubated with a primary antibody at 4°C overnight and then incubated with a secondary antibody at room temperature for 1 h. Details regarding primary and secondary antibodies are listed in Table 3 . The blotted proteins were quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). β-Actin was set as an internal control. The relative expression level of each target protein was displayed as a ratio of target protein/β-actin protein. All the assays were performed at least in triplicate independently.
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