The relative protein expressions of MRP, LRP, BCRP, Bcl2, and PKC-α in HepG2 and HepG2/DOX cells and tumor tissues were detected by western blotting. Total proteins in cells or tumor tissues were extracted by RIPA buffer (Beyotime Biotechnology, Shanghai, China), and then the protein concentration was detected using a bicinchoninic acid (BCA) assay kit (Solarbio, USA). After 20-μg protein samples were separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes, the membranes were blocked with 5% nonfat milk and washed with Tris-buffered saline containing Tween 20 (TBST). The membranes were incubated with the primary antibodies against MRP (DF8801, Affinity), LRP1 (ab92544, Abcam), BCRP (ab207732, Abcam), Bcl2 (ab196495, Abcam), PKC-α (ab32376, Abcam), and β-actin (ab8226, Abcam) overnight at 4°C. The membranes were then washed with TBST and further incubated with the horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature. Finally, the protein bands were exposed to the chemiluminescent reagent (enhanced chemiluminescence) for 3 min and captured with the Chemi Capture software.
Ab207732
Manufactured by Abcam
Sourced in United States
Ab207732 is a recombinant monoclonal antibody that recognizes the protein GAPDH. The antibody is produced in HEK293 cells and purified using protein A affinity chromatography. The antibody is suitable for use in applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab170904, by Abcam (3 mentions)
Ab196495, by Abcam (2 mentions)
Ab233383, by Abcam (2 mentions)
Ab109250, by Abcam (2 mentions)
Ab92544, by Abcam (1 mentions)
Ab32376, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Bca assay kit, by Solarbio (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab52492, by Abcam (1 mentions)
Fl 335, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Labrasol, by Gattefosse (1 mentions)
Capryol 90, by Gattefosse (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab267373, by Abcam (1 mentions)
Ab273093, by Abcam (1 mentions)
Ab92744, by Abcam (1 mentions)
Ab53154, by Abcam (1 mentions)
9 protocols using ab207732
1
Protein Expression Analysis in HepG2 Cells
2
Evaluating Stem Cell Markers in Cells
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Protein extracts from cells or tumour tissues were mixed with loading buffer, heated at 70°C for 10 minutes, separated on SDS‐PAGE gels and transferred to polyvinylidene fluoride (Millipore) membranes. Membranes were blocked for 2 hours in 5% bovine serum albumin and incubated overnight at 4°C with SP rabbit polyclonal antibodies against SOX2 (H‐65; sc‐20088, Santa Cruz, 1/1000), OCT4 (H‐134; sc‐9081, Santa Cruz, 1/1000), ABCG2 (B‐25; sc‐130933, Santa Cruz, 1/1000), JAMA (H202‐106; sc‐59845, Santa Cruz, 1/1000), Akt (H‐136; sc‐8312, Santa Cruz, 1/1000), ALDH1 (ab52492, Abcam, 1/1000), ABCG2 (ab207732, Abcam, 1/1000) and GAPDH (FL‐335; sc‐25788, Santa Cruz, 1/1000). Membranes were then incubated with horseradish peroxidase (HRP)–conjugated secondary antibody (sc‐2004, Santa Cruz, 1:1000) for 1 hour at room temperature. Finally, bands were visualized using enhanced chemiluminescence (Thermo Scientific Pierce).
3
Efflux Transporters Expression Analysis
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HupA was supplied by MULTI SCIENCES (Hangzhou, China). Propylene glycol monocaprylate (Capryol 90) and caprylocaproyl macrogolglycerides (Labrasol) were purchased from Gattefossé (Saint-Priest, France). Propan-2-yl tetradecanoate (IPM) was provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Anti-P-gp antibody [EPR10364] (ab168337), anti-BCRP/ABCG2 antibody [EPR20080] (ab207732), anti-MRP1 antibody [EPR21062] (ab233383), and goat anti-rabbit IgG (H+L)-horseradish peroxidase-conjugated (ab181448) secondary antibody were purchased from Abcam (Cambridge, UK). The water-quenching near infrared fluorescent probe P2 (λabs/λem = 720 nm/740 nm) was provided by the School of Pharmacy, Fudan University.
4
Protein Expression Profiling Protocol
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Total protein was extracted, separated and transferred to membranes, which were then blocked with 5% skim milk. Next, the protein‐loaded membrane was probed with antibodies against APE1 (ab92744, 1:500, Abcam), MDR1 (ab170904, 1:500), MRP (ab261871, 1:500), LRP (ab273093, 1:500), ABCG2 (ab207732, 1:500), STAT3 (ab68153, 1:500, Abcam), p‐STAT3 (ab267373, 1:1000, Abcam), Bcl‐2 (ab196495, 1:1000, Abcam), Bax (ab53154, 1:500, Abcam), cleaved caspase‐3 (ab2302, 1:1000, Abcam) and β‐actin (ab8227, 1:500, Abcam) and then with HRP‐labelled anti‐rabbit IgG secondary antibodies (No. 7074, 1:5000; CST, Danvers, MA) at 37°C for 1 h. The blots were visualized via an ECL reagent (32106, Thermo Fisher Scientific), normalized to β‐actin.
5
Western Blot Analysis of Protein Markers
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Total protein was extracted from the frozen cells using RIP assay (RIPA) buffer. Thirty micrograms of protein was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a polyvinylidene difluoride membrane (PVDF), the proteins were blocked with 5% non-fat milk and incubated in primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody for 2 h. The targeted proteins were detected and visualized with an enhanced chemiluminescence system (ECL, Beyotime, Shanghai, China) and X-ray film (GE Healthcare). β-actin was used as a loading control. The antibodies used (all purchased from Abcam [Cambridge, MA, USA]) are listed as follows: CD63 (ab134045), Hsp70 (ab2787), TSG101 (ab125011), SOX4 (ab70598), Oct4 (ab200834), NANOG (ab109250), ABCG2 (ab207732), β-actin (ab8227), PKM2 (ab137852), HiF-1α (ab179483), GLUT1 (ab115730), HK2 (ab209847), goat anti-rabbit immunoglobulin G (IgG) H&L (HRP) (ab6721), and rabbit anti-mouse IgG H&L (HRP) (ab6728).
6
Protein Extraction and Western Blot Analysis
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RIPA (Beyotime, China) buffer supplemented with phosphatase inhibitor (Beyotime) and a Nuclear Protein Extraction Kit (Beyotime) were used to extract total and nuclear proteins from NSCLC cells. The extracted proteins were transferred to PVDF membranes after fractionation by SDS-PAGE. After blocking in 5% milk for 2 h, the PVDF membranes were incubated at 4 °C overnight with a specific antibody. Protein bands were visualized using ExPlus ECL (ZOMANBIO) with a MicroChemi system (DNR, Israel). The primary antibody against glycogen synthase kinase 3 beta (GSK3β) (12456 T) was purchased from Cell Signaling Technology (USA), and primary antibodies against FOXC1 (ab5079), LMNB1 (ab133741), ABCG2 (ab207732), SOX2 (ab92494), Oct4 (ab181557), phospho-GSK3β (pGSK3β) (ab75814) and NANOG (ab109250) were purchased from Abcam (USA). The primary antibody against GAPDH (60004–1-Ig) was purchased from Proteintech (China), and the primary antibody against beta-actin (A01011–1) was purchased from Abbkine (China).
7
Western Blot Analysis of Stem Cell Markers
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Protein was extracted from cells with a protein lysis buffer (Beyotime). Total proteins were quantitated with a BCA assay kit (Pierce, Rockford, IL, USA), followed by separation with 10% SDS-PAGE gels. Subsequently, the proteins were removed from gel to PVDF membrane and then blocked with 5% milk for 1 h. After incubation with primary antibody overnight at 4 °C, the membranes were incubated by HRP-conjugated secondary antibody for 2 h. Lastly, the blots were measured by ECL substrate. The primary antibodies included: anti-CD133 (130–092–395, 1:100, Miltenyi Biotec), anti-CD151 (SAB1402716, 1:50, Sigma), anti-E2F1 (ab4070, 1:500; Abcam), anti-MDR1 (ab170904, 1:100, Abcam), anti-BCRP1 (ab207732, 1:100, Abcam), anti-γ-H2AX (ab81299, 1:400; Abcam), anti-H2AX (ab11175, 1:200, Abcam), anti-AKT (#9272, 1:1000, Cell Signaling, Danvers, MA, USA), anti-p-AKT (#9271, 1:1000, Cell Signaling), anti-mTOR (ab134903, 1:100, Abcam), anti-p-mTOR (#2971, 1:100, Cell Signaling), anti-cyclinD1 (#2922, 1:1000, Cell Signaling) and anti-β-actin (A1978, 1:6000, Sigma).
8
Western Blot Analysis of ABCG2, METTL3, and GAPDH
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The Western blot assay was conducted as previously described [22 (link)]. Cells and tissues were lysed in RIPA buffer (Cat.9806, Cell Signaling Technology, Danvers, MA, USA) containing phosphatase inhibitors (Cat.P2850, Sigma, St. Louis, MO, USA) and a protease inhibitor cocktail (Cat.P8340, Roche, Basel, CH). The lysate was subjected to SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and incubated with primary antibody, followed by HRP conjugated secondary antibody. The band was visualized using SuperSignal™ West Pico PLUS (Cat.34580, Invitrogen). The primary antibodies used are anti-ABCG2 (1:1000 dilution, ab207732, Abcam), anti-METTL3 (1:2000 dilution, ab195352, Abcam), and anti-GAPDH (1:15000 dilution, ab181602, Abcam).
9
Quantitative Protein Expression Analysis
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The proteins were extracted from cells, and were measured concentrations using a BCA protein assay kit (Beyotime Biotec, China). Protein samples were fractionated using 4–20% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, NY, USA). After 1 h blocking with 5% non-fat milk at room temperature, the membranes were then incubated for 12 h at 4 °C with rabbit anti-human primary antibodies: E2F1 (#3742, CST), RB (#9313, CST), pRB (#8516, CST), PCNA (#13110, CST), c-myc (#5605, CST), pAKT (#4060, CST), CCND1 (ab134175, Abcam), CCNE1 (ab33911, Abcam), CCNA2 (ab181591, Abcam), MDR1 (ab170904, Abcam), MRP1 (ab233383, Abcam), BCRP (ab207732, Abcam), AKT (db1607, Diagbio), Bcl-2 (db2374, Diagbio), Bcl-xl (db225, Diagbio), Bax (db819, Diagbio), GAPDH (db106, Diagbio), and Tubulin (AC015, ABclonal) were used as endogenous controls. The proteins were detected by ECL detection solution (Thermo Scientific™) and analyzed by Image Lab software (Bio-Rad).
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