The experiment was conducted as previously described [22 (link)]. The cells were lysed in Radioimmunoprecipitation Assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). After the total protein was collected, bicinchoninic acid (BCA) protein reagent kit (ThermoFisher) was employed for the measurement of protein concentration. The samples electrophoresed through the employment of 10% sodium dodecyl sulfate–polycrylamide gel electrophoresis (SDS-PAGE) were shifted to polyvinylindene difluoride (PVDF) membranes. Subsequently, the membranes were sealed with 5% defatted milk for 1 h and cultured with specific primary antibodies to MITF, MMP2, MMP7, MMP9, E-cadherin, N-cadherin, and GAPDH. Antibodies were obtained from Abcam (Cambridge, UK) overnight at 4°C. Subsequently, the secondary antibodies were incubated with these membranes for 2 h. The western blots were captured and scanned via BioImaging Systems (BIO-RAD, CA, USA).
Bca protein reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA protein reagent kit is a colorimetric assay used for the quantitative determination of total protein concentration. It utilizes the bicinchoninic acid (BCA) reaction to measure the reduction of copper (Cu2+) to copper (Cu1+) by protein in an alkaline medium. The resulting purple-colored reaction is measured at 562 nm, and the protein concentration is determined by comparison to a standard curve.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Mini protease inhibitor cocktail, by Roche (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab225958, by Abcam (1 mentions)
Biospectrum 600 imaging system, by Analytik Jena (1 mentions)
Ab8227, by Abcam (1 mentions)
Ecl plus kit, by Beyotime (1 mentions)
7 protocols using bca protein reagent kit
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Signaling Proteins
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The cells were harvested and lysed using RIPA lysis buffer containing configure RIPA, PIC, and PMSF at a ratio of 100:2:1 (Beyotime Biotechnology, Shanghai, China). The total protein was extracted and the protein concentration was detected by BCA protein reagent kit (Thermofisher Scientific, Inc). The samples were electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% defatted milk in TBST for 1 h at room temperature and then incubated with specific primary antibodies overnight at 4 °C (Table 1 ). Subsequently, the HRP-conjugated secondary antibodies were incubated at room temperature for 1.5 h. The protein bands were detected and scanned by BioImaging Systems (BIO-RAD, CA, USA). Anti-GAPDH antibody was used as an internal control to normalize protein levels.
The antibodies used in the western blotting analysis
| Protein | Producer | Catalog number | Dilution |
|---|---|---|---|
| PGC-1β | NOVUS | NB110-58858G | 1:500 |
| FOXA2 | Abcam | ab108422 | 1:1000 |
| GAPDH | Abcam | ab128915 | 1:5000 |
| p-AKT | Abcam | ab38449 | 1:500 |
| AKT | CST | #9272 | 1:1000 |
| PI3K | CST | #4257 | 1:500 |
| mTOR | CST | #2983 | 1:500 |
| Rictor | CST | #2114 | 1:1000 |
| Raptor | CST | #2280 | 1:1000 |
| PDK1 | CST | #3062 | 1:1000 |
CST cell signaling technology
3
Western Blot Protein Quantification
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The total protein of each group was extracted and the protein concentration was detected by the BCA protein reagent kit (Thermo Fisher Scientific, Inc.). The protein samples were separated with 10% SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 5% defatted milk in TBST at 25°C for 1 h. The membrane was incubated with a specific primary antibody (1 : 1 000) at 4°C overnight. Then, the HRP-conjugated secondary antibodies were incubated at 25 °C for 2 h. After washing the membrane with TBST 3 times, the protein bands were detected and scanned by Bio Imaging Systems (BIO-RAD, CA, USA). The absorbance of the protein bands in each group was measured by Quantity One gel analysis software. The protein expression level is assessed by the ratio of the target band to the GAPDH band. Each protein sample was applied with 3 repeats.
4
Quantification of Amyloid-Beta Peptides
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The right brain tissue was homogenized and sonicated at 4°C in RIPA lysis buffer (Thermo Scientific™, cat. no. 89900) mixed with Mini Protease Inhibitor Cocktail (Roche, cat. no.04693124001). The supernatants were collected and quantified for the concentrations of total protein by using a bicinchoninic acid (BCA) protein reagent kit (Thermo Scientific™, cat. no. 23235). And Aβ1-40 and Aβ1-42 were individually measured by ELISA kits (R&D Systems, cat. no. DAB140B and cat. no. DAB142, respectively) according to the manufacturer’s protocols.
5
Protein Expression Analysis of SMAD2
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First, each group of cells was harvested, and then proteins were extracted using a lysis buffer. Next, we quantified the proteins with a BCA protein reagent kit (Thermo Scientific, USA). Equal amounts of protein extractions were isolated by SDS‐PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes at 300 mA for 90 min and incubated overnight with primary antibodies at 4°C (1:1000; Abcam, Cambridge, MA, USA) against SMAD2 (ab225958) and β‐actin (ab8227). Last, we incubated proteins with the second antibodies (Abcam, ab205718) and analyzed proteins with the ECL plus kit (Beyotime, Haimen, China, P0018S). Images were gained with a BioSpectrum 600 Imaging System (UVP, CA, USA). This experiment was repeated three times and the average value was taken.
6
Cellular Sesamol Uptake Kinetics
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The cells were exposed to 1 mM (200 µL) sesamol and incubated at 37 °C at various times (0.5, 1, 3, 5, 10, and 15 min). The media was plated into 96-well plates prior to measuring the extracellular sesamol content by HPLC analysis. The cells were washed twice with ice-cold PBS, solubilized by methanol, and centrifuged at 2400× g, at 4 °C for 20 min. The precipitated protein was re-solubilized by 0.1 M NaOH and 25 µL was taken for protein determination using a BCA Protein Thermo Fisher Scientific Reagent Kit (Pierce Biotechnology, Rockford, IL, USA). The sesamol content in 20 µL of cell lysate was quantified using HPLC assay as described in the analytical procedure.
The cellular uptake of sesamol was determined based on the sesamol content in cells (nmol/mg protein) to medium (nmol/µL) (expressed as C/M ratio, µL/mg protein). The linear regression of the C/M ratio from 0.5 to 3 min was performed (r2 > 0.90); to give the initial distribution volume (µL/mg protein) from the y-intercept, and the initial uptake clearance (µL/min/mg protein) from the slope.
The cellular uptake of sesamol was determined based on the sesamol content in cells (nmol/mg protein) to medium (nmol/µL) (expressed as C/M ratio, µL/mg protein). The linear regression of the C/M ratio from 0.5 to 3 min was performed (r2 > 0.90); to give the initial distribution volume (µL/mg protein) from the y-intercept, and the initial uptake clearance (µL/min/mg protein) from the slope.
7
Quantification of Intracellular Sesamol
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SK-MEL-2 cells were seeded into 24-well plates (2 × 105 cells per well) and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. The cell medium was removed, and the cells were pre-incubated with prewarmed (37 °C) Hank’s Balanced Salt Solution (HBSS) pH 7.4 for 20 min. The cells were treated with a sesamol working solution (final concentration of 1 mM) and incubated at 37 °C for 5, 10, 20, 30, and 45 min. At specified intervals, the cells were washed twice with ice-cold (4 °C) PBS and lysed with 200 µL of methanol for 15 min at room temperature. Proteins were precipitated by centrifugation at 2400× g at 4 °C for 20 min. The intracellular sesamol concentration was quantified using HPLC, as described above (Section 3.5 ). The precipitated proteins were resolubilized with 0.1 M sodium hydroxide. The solubilized protein was pipetted for protein determination using a BCA Protein Thermo Fisher Scientific Reagent Kit (Pierce Biotechnology, Rockford, IL, USA). Intracellular unbound sesamol concentrations are presented in nanomoles per milligram (nmol/mg protein).
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