Total proteins were extracted from HepG2 cells and Huh7 cells using radio-immunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China). After protein quantitation using the bicinchoninic acid assay, the proteins were separated by PAGE with a 5% stacking gel and a 10% separating gel and then transferred onto a nitrocellulose membrane using wet transfer apparatus. Next, the membrane was blocked with 5% BSA for 1 h and incubated with the diluted rabbit anti-human antibodies from Abcam (Cambridge, UK) at 4°C overnight: MDR-1 (ab129450, 1:1,000), MRP2 (ab203397, 1:500), MRP3 (ab107083, 1:250), GAPDH (ab37168, 1 μg/mL), and E-cadherin (ab15148, 1:500). Next, the membrane was washed using PBS/Tween-20 and further incubated by applying 5% skimmed milk-diluted secondary antibody goat anti-rabbit IgG (ab205718, 1: 5000) for 1 h in avoidance of light. Subsequently, the protein bands were developed and visualized in a gel imaging system (MG8600, Bio-Rad, Hercules, CA, USA). Images were analyzed using the Image-Pro Plus software (Version 7.0, Media Cybernetics, Silver Springs, MD, USA). The expression levels of target proteins relative to GAPDH were calculated.
Ab203397
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab203397 is a laboratory equipment product. It is designed for use in scientific research and analysis. The core function of this product is to [core function description]. No further details or interpretation on the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab15148, by Abcam (1 mentions)
Ab129450, by Abcam (1 mentions)
Rabbit anti human antibodies, by Abcam (1 mentions)
Mg8600, by Bio-Rad (1 mentions)
Ab37168, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Ab89443, by Abcam (1 mentions)
Ab1431, by Abcam (1 mentions)
Ab243153, by Abcam (1 mentions)
Ab9643, by Abcam (1 mentions)
Wl01637, by Wanlei (1 mentions)
Wl01372, by Wanlei (1 mentions)
Pc0020, by Solarbio (1 mentions)
Ab129815, by Abcam (1 mentions)
Ab84400, by Abcam (1 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
4 protocols using ab203397
1
Quantification of Drug-resistance Proteins
2
Renal Protein Extraction and Western Blot
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Proteins were extracted from renal tissue using an ice-cold lysis buffer, and the protein concentrations were quantified using a BCA assay kit (PC0020; Solarbio). SDS-PAGE was used to separate equal amounts of protein. The blocked membrane was incubated using anti-Bax (1 : 500; WL01637, WanleiBio), anti-P-p53 (1 : 2,000; ab1431, Abcam), anti-PUMA (1 : 3,000; ab9643, Abcam), anti-caspase-3 (1 : 2,000; 9661, CST), anti-Nrf2 (1 : 500; ab89443, Abcam), anti-HO-1 (1 : 500; WL01637, WanleiBio), anti-OCT2 (1 : 500; ab243153, Abcam), anti-MRP2 (1 : 500; ab203397, Abcam), and anti-β-actin (1 : 1,000; WL01372, WanleiBio) antibodies overnight at 4°C. Then, the membranes were incubated with a suitable horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. Finally, an ECL western blotting substrate was used to visualize the immunoblots.
3
Western Blot Analysis of Stem Cell Markers
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Cells were treated with RIPA Lysis Buffer (Cat #P0013B, Beyotime, Beijing, China), followed by determining protein concentration using Bradford Protein Assay Kit (Cat #P0006, Beyotime). Then 30 μg protein was separated by 10% SDS-PAGE. The protein was transferred onto PVDF membranes (Bio-Rad). Blocking was carried out with 10% non-fat milk for 1.5 hours at 37°C. Then the membranes were washed with TBST for 15 minutes, 3 times, and incubated with the primary antibodies at 4°C overnight. The primary antibodies against ALDH1 (ab129815), Nanog (ab80892), ABCG2 (ab203397), MDR1 (ab3366), p-mTOR (ab84400), cleaved caspase-3 (ab32042), and cleaved PARP (ab32064) were purchased from Abcam. The primary antibodies against β-actin (Cat #60008-1-Ig), p-Akt (Cat #66444-1-Ig), Akt (Cat #10176-2-AP), caspase-3 (Cat #19677-1-AP), PARP (Cat #13371-1-AP), E-cadherin (Cat #20874-1-AP), and vimentin (Cat #10366-1-AP) were purchased from Proteintech. Then membranes were washed with TBST for 15 minutes, 3 times, followed by incubating with HRP-labeled goat anti-rabbit IgG(H+L) (Cat #A0208, Beyotime), or HRP-labeled goat anti-mouse IgG(H+L) (Cat #A0216, Beyotime). ECL Plus (Cat #PE0010, Solarbio) was used to detect chemiluminescent signals in Bio-Rad ChemiDoc™ Touch (CM002554, Bio-Rad).
4
Quantification of Hepatic Transporter Proteins
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Protein expression of Mrp2, Bsep, and P-gp was measured as previously described [21 (link)]. Briefly, total protein was obtained by homogenizing 100 mg of liver samples with equal volume of a tissue lysis buffer. Protein samples (30–50 µg) were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–15% gradient gel (Bio-Rad). Separated proteins were transferred onto a Potran 0.45 μm nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween20 (TBST) (Biosesang, Seoul, Republic of Korea) for 1 h and incubated with primary antibody at 4 °C for 12 h. Primary antibodies were used as follows: anti-Mrp2 antibody (ab203397, dilution 1:1000, Abcam, San Francisco, CA, USA), anti-Bsep antibody (ab217532, dilution 1:1000, Abcam), anti-P-gp antibody (E1Y7S, dilution 1:1000, Cell signaling technology, Danvers, MA, USA) and beta-actin antibody (1:1000, Cell signaling technology). The membrane was rinsed twice with TBST at 25 °C and treated with horseradish peroxidase-labeled anti-rabbit IgG antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using a Luminata Forte enhanced chemiluminescence system (Millipore, Burlington, MA, USA), and beta-actin served as the loading control.
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