After indicated treatment, the cells were lysed by RIPA lysis buffer (Cell Signaling Technology) for 30 min on ice. The 50 g protein sample was subjected to 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked by 5% non-fat milk for 1 h at room temperature. The membranes then were incubated with primary antibodies (anti-MDR, cat no. sc-55510, 1:1000, Santa Cruz Biotechnology; anti-P-gp, cat no. ab103477, 1:1000, Abcam; anti-TCF4, cat no. sc-166699, 1:1000, Santa Cruz Biotechnology; anti-GLUT3, cat no. sc-74399, 1:1000, Santa Cruz Biotechnology; and anti-β-actin, cat no. sc-47778, 1:1000, Santa Cruz Biotechnology) overnight at 4°C. The membranes were washed three times with PBS. After washing, the membranes were incubated with appropriate secondary antibodies for 1 h at 37°C. The bands were visualized by enhanced chemiluminescence.
Anti p gp
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-P-gp is a laboratory reagent used for the detection and quantification of P-glycoprotein (P-gp), a transmembrane protein involved in drug efflux. It can be used in a variety of cell-based assays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti p53, by Abcam (2 mentions)
Anti ciapin1, by Abcam (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti mrp2, by Abcam (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Anti ahr, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Chemiluminescence kit, by Beyotime (2 mentions)
Anti c myc, by Abcam (2 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Anti tcf4, by Santa Cruz Biotechnology (1 mentions)
Anti glut3, by Santa Cruz Biotechnology (1 mentions)
Anti bmi1, by Abcam (1 mentions)
Gapdh, by Proteintech (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
20 protocols using anti p gp
1
Protein Expression Profile Analysis
2
Western Blot Analysis of BMI1, P-GP, and p53
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WB was carried out base on the standard protocol, as mentioned earlier [37 (link)]. The primary antibodies were applied as followed: anti-BMI1 (Abcam, Cat. No. ab126783), anti-P-GP (Abcam, Cat. No. ab170904) and anti-p53 (Abcam, Cat. No. ab1101). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Proteintech, Cat. No. 60004-1-Ig) was used as a loading control.
3
Western Blot Analysis of P-glycoprotein in Rat Brain
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The rat brain vessels were lysed in radioimmunoprecipitation assay buffer containing protease inhibitor (150 mM Sodium chloride, 0.1% SDS, 1% Triton X-100, 1% Sodium deoxycholate, and 50 mM Tris–Hcl [pH 7.5, 2 mM EDTA]). After centrifugation at 16000 g for 20 min, the protein concentration was determined using PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States). The proteins (30 μg/lane) were separated using a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, United States). Membranes were blocked with 5% non-fat skim milk in phosphate buffered saline containing 0.05% tween 20 for 1 h at room temperature (RT) and were incubated overnight at 4°C with rabbit monoclonal anti-P-gp (Abcam, Cambridge, MA, United States) or anti-β-actin antibodies (Abcam, Cambridge, MA, United States), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (Abcam, Cambridge, MA, United States) for 2 h at RT. The signal was detected using an ECL plus chemiluminescence kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, United States). The band density was quantified by ImageJ software (1.52v, National Institutes of Health, Bethesda, MD, United States) (Yang and Rosenberg, 2011 (link)).
4
Protein Extraction and Immunoblotting Protocol
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Tissue samples were split, and protein extraction was conducted with Tissue Protein
Extraction Reagent (T-PER, 78510, Pierce, USA) in accordance with the instruction
manual. Briefly, tissue samples were washed twice with DPBS and then were mixed with
T-PER at a ratio of 1:20. The lysates were centrifuged at 10,000 gfor 5 min and supernatants were collected. The concentration of total protein in the
supernatant was measured by bicinchoninic acid assay and adjusted to a concentration
of 10 μg/µL. Immunoblotting was conducted as previously described (13 (link)). The following antibodies were used:
anti-CIAPIN1 (1:500, Abcam, UK), anti-P53 (1:300, Santa Cruz, USA), anti-P-gp (1:100,
Abcam), anti-β-actin (1:1000, Santa Cruz), anti-mouse horseradish peroxidase
(HRP)-conjugated antibody (1:5000, Santa Cruz) and anti-rabbit HRP-conjugated
antibody (1:3000, Cell Signaling Technology, USA).
Extraction Reagent (T-PER, 78510, Pierce, USA) in accordance with the instruction
manual. Briefly, tissue samples were washed twice with DPBS and then were mixed with
T-PER at a ratio of 1:20. The lysates were centrifuged at 10,000 gfor 5 min and supernatants were collected. The concentration of total protein in the
supernatant was measured by bicinchoninic acid assay and adjusted to a concentration
of 10 μg/µL. Immunoblotting was conducted as previously described (13 (link)). The following antibodies were used:
anti-CIAPIN1 (1:500, Abcam, UK), anti-P53 (1:300, Santa Cruz, USA), anti-P-gp (1:100,
Abcam), anti-β-actin (1:1000, Santa Cruz), anti-mouse horseradish peroxidase
(HRP)-conjugated antibody (1:5000, Santa Cruz) and anti-rabbit HRP-conjugated
antibody (1:3000, Cell Signaling Technology, USA).
5
Molecular Mechanisms in HepG2 Cells
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HepG2 and HepG2/ADR cells were harvested after 48 h treatment and lysed using RIPA buffer (R0278; Sigma Aldrich, St Louis, MO, USA). The lysates were centrifuged and the supernatant was collected for Western blotting as the routine protocol. The following antibodies were used: anti-AEG-1 (1:2,000; ProteinTech, IL, USA), anti-YAP1 (1:1,000; Cell signaling, MA, USA), anti-Bcl-2 (1:1,000; Cell signaling, USA), anti-cleaved-caspase-3 (1:1,000; Cell signaling, USA), anti-caspase-3 (1:1,000; Cell signaling, USA), anti-Bax (1:1,000; Cell signaling, USA), anti-P-gp (1:1,000; Abcam, Cambridge, UK), and anti-α-tubulin (1:1,000; Promoter, Wuhan, China).
6
Western Blot Analysis of P-glycoprotein
7
Plasmid Transfection and Antibody Analysis
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The following plasmids were used for transfection: pcDNA3.1(-)-GFP-LC3 [47 (link)], mRFP-GFP-LC3 construct [48 (link)]. hPC1-expressing plasmid WT (REP10) was a gift from Gregory Germino (Addgene plasmid #21368) [49 (link)] as was hPC2-expressing plasmid (M-PKD2 (OF2-3); Addgene plasmid #21370) [21 (link)].
The following siRNAs were used: siRNA against mouse Pkd2 (AM16708, assay ID 150154, Thermo Fischer Scientific), siRNA against mouse BECN1 (4390771, Ambion, Woolston, UK) and negative control DsiRNA (51-01-14-04, IDT).
The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-β-Actin (4970), anti-FoxO1 (14952), anti-ULK1 (8054), anti-phospho-ULK1 (Ser555) (5869), anti-phospho-ULK1 (Ser757) (14202), anti-phospho-ULK1 (Ser317) (3776), anti-AMPK (5832), anti-phospho AMPK (Thr172) (50081), anti-S6 (2217) and anti-p-S6 (4858). Anti-PC1 (sc-130554), anti-PC2 (sc-28331) and anti-SV40 LT were from Santa Cruz Biotechnology (Dallas, TX, USA), anti-Sqstm1 (P0067) and anti-EpCam (HPA026761) from Sigma-Aldrich (Saint-Louis, MO, USA, anti-LC3 from Nanotools (clone 5F10; München, Germany), anti-PgP from Abcam (ab170904; Cambridge, UK) and anti-AQP1 from Novus Biologicals (84488; Abingdon, UK). Secondary anti-rabbit and anti-mouse antibodies were from Bio-Rad Laboratories (Hercules, CA, USA).
The following siRNAs were used: siRNA against mouse Pkd2 (AM16708, assay ID 150154, Thermo Fischer Scientific), siRNA against mouse BECN1 (4390771, Ambion, Woolston, UK) and negative control DsiRNA (51-01-14-04, IDT).
The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-β-Actin (4970), anti-FoxO1 (14952), anti-ULK1 (8054), anti-phospho-ULK1 (Ser555) (5869), anti-phospho-ULK1 (Ser757) (14202), anti-phospho-ULK1 (Ser317) (3776), anti-AMPK (5832), anti-phospho AMPK (Thr172) (50081), anti-S6 (2217) and anti-p-S6 (4858). Anti-PC1 (sc-130554), anti-PC2 (sc-28331) and anti-SV40 LT were from Santa Cruz Biotechnology (Dallas, TX, USA), anti-Sqstm1 (P0067) and anti-EpCam (HPA026761) from Sigma-Aldrich (Saint-Louis, MO, USA, anti-LC3 from Nanotools (clone 5F10; München, Germany), anti-PgP from Abcam (ab170904; Cambridge, UK) and anti-AQP1 from Novus Biologicals (84488; Abingdon, UK). Secondary anti-rabbit and anti-mouse antibodies were from Bio-Rad Laboratories (Hercules, CA, USA).
8
Immunohistochemical Antibody Panel for Cancer
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The mouse monoclonal antibodies used in this study were: anti-MUC1 (clone BSB-44, Bio SB, dilution 1:50), anti-CEA (clone BSB-31, Bio SB, prediluted), anti-CA19-9 (clone 121SLE, dilution 1:75), anti-MRP2 (clone M2III-6, dilution 1:100), anti-MRP3 (clone M3II-9, dilution 1:5), anti-Pg-P (clone JSB-1, dilution 1:5), anti-TIM3 (clone TIM3/3113, dilution 1:50), anti-PD-L1 (clone ABM4E54, dilution 1:500), anti-HLA-ABC (clone EMR8-5, dilution 1:50), anti-IL-8 (clone 807, dilution 1:50) (Abcam, Cambridge, UK), anti-survivin (clone 8E2, dilution 1:30), anti-TNF-a (clone F6C5, dilution 1:50), and anti-TGF-b (clone TB21, dilution 1:50) (ThermoFisher Scientific, Waltham, MA, USA). The rabbit polyclonal antibodies used were: anti-A2aR (PA-33323, ThermoFisher Scientific, dilution 1:200), anti-IFN-g (ab25101, dilution 1:500), anti-CXCR4 (p-S339) (ab74012, dilution 1:75), anti-CCR7 (ab140758, dilution 14 mg/mL), and anti-IL-6 (ab6672, dilution 1:600) (from Abcam).
9
Protein Expression Analysis in Prostate Cancer Cells
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LNCaP and VCaP cells were lysed using radioimmunoprecipitation assay buffer (Beyotime, China). Protease inhibitors (Beyotime, China) was added into the lyse buffer to avoid protein degradation. Samples (20 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then samples were transferred onto nitrocellulose membranes. After that, samples were incubated with following primary antibodies: anti-AhR (1:500, Abcam, UK), anti-P-gp (1:1000, Abcam, UK), anti-β-actin (1:1000, Abcam, UK) and anti-c-Myc (1800, Abcam, UK). chemiluminescence kit (Beyotime, China) were used for protein detection. Each experiment was repeated for three independent times.
10
Western Blot Analysis of Drug Transporters
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Western blot analysis was performed using a previously reported method [34 (link)]. The homogenates prepared from the mucosa and liver or mucosa microsomes of each group with equal amounts of protein were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. After blocking, the membranes were hybridized with antibodies against anti-P-gp (Abcam, Cambridge, UK), anti-OATP2B1 (GeneTex Inc, Irvine, CA, USA), anti-OATP2 (Merck Millipore, MA, USA), and anti-CYP3A1 (Calbiochem, Darmstadt, Germany) at 4 °C with gentle agitation overnight. After washing twice with PBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, Cambridge, UK) at room temperature for 1 h. Equal loading across the lanes was confirmed by staining the blot with Ponceau S solution.
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