- MA5-13854 - Invitrogen Antibodies ABCB1 antibody

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Chai AB, Callaghan R, Gelissen IC
International journal of molecular sciences 2022 Jan 18;23(3)

Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells.

Kobori T, Tameishi M, Tanaka C, Urashima Y, Obata T
PloS one 2021;16(5):e0250889

LightSpot(®)-FL-1 Fluorescent Probe: An Innovative Tool for Cancer Drug Resistance Analysis by Direct Detection and Quantification of the P-glycoprotein (P-gp) on Monolayer Culture and Spheroid Triple Negative Breast Cancer Models.

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Ouyang X, Dong C, Ubogu EE
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Combined effects of octreotide and cisplatin on the proliferation of side population cells from anaplastic thyroid cancer cell lines.

Li Z, Jiang X, Chen P, Wu X, Duan A, Qin Y
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The Human Blood-Nerve Barrier Transcriptome.

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Influence of P-glycoprotein on brucine transport at the in vitro blood-brain barrier.

Xu DH, Yan M, Li HD, Fang PF, Liu YW
European journal of pharmacology 2012 Sep 5;690(1-3):68-76

ABCB1 is predominantly expressed in human fetal neural stem/progenitor cells at an early development stage.

Yamamoto A, Shofuda T, Islam MO, Nakamura Y, Yamasaki M, Okano H, Kanemura Y
Journal of neuroscience research 2009 Sep;87(12):2615-23

[The effect of resistance-related proteins on the prognosis and survival of patients with osteosarcoma: an immunohistochemical analysis].

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Acta orthopaedica et traumatologica turcica 2009 Jan-Feb;43(1):28-34

Effect of the novel antipsychotic drug perospirone on P-glycoprotein function and expression in Caco-2 cells.

Zhou YG, Li KY, Li HD
European journal of clinical pharmacology 2008 Jul;64(7):697-703

Efficacy of Hsp90 inhibition for induction of apoptosis and inhibition of growth in cervical carcinoma cells in vitro and in vivo.

Schwock J, Pham NA, Cao MP, Hedley DW
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Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells.

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Guan J, Chen XP, Zhu H, Luo SF, Cao B, Ding L
World journal of gastroenterology 2004 Dec 1;10(23):3522-7

Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line.

Guan J, Chen XP, Zhu H, Luo SF, Cao B, Ding L
World journal of gastroenterology 2004 Dec 1;10(23):3522-7

P-glycoprotein expression and distribution in the rat placenta during pregnancy.

Novotna M, Libra A, Kopecky M, Pavek P, Fendrich Z, Semecky V, Staud F
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Examination of the functional activity of P-glycoprotein in the rat placental barrier using rhodamine 123.

Pavek P, Staud F, Fendrich Z, Sklenarova H, Libra A, Novotna M, Kopecky M, Nobilis M, Semecky V
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FEBS letters 1999 Jan 15;442(2-3):208-14

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of p170 was performed by loading 25 µg of HepG2 (lane 1), 293T (lane 2) and mouse liver (lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a p170 monoclonal antibody (Product # MA5-13854) at a dilution of 1:100 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~170 kDa in HepG2 and 293T cells.

Supportive validation

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Experimental details: Knockout of ABCB1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR634315_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of ABCB1 was performed by loading 30 µg of Caco-2 wild type (Lane 1) and Caco-2 ABCB1 KO (Lane 2) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with P-Glycoprotein Monoclonal Antibody (F4) (Product # MA513854, 1:200 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 dilution) using the iBright™ FL1500 (Product # A44115). Chemiluminescent detection was performed usingSuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to ABCB1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of p170 (green) showing staining in the membrane of A431 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a p170 monoclonal antibody (Product # MA5-13854) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of p170 in CHO cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a p170 monoclonal antibody (Product # MA5-13854) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of p170 in Jurkat cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a p170 monoclonal antibody (Product # MA5-13854) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of p170 in Raji cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a p170 monoclonal antibody (Product # MA5-13854) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 1 Gene expression pattern and subcellular localization of ezrin, radixin, and moesin (ERM) and P-gp in LS180 cells. (A, B) Representative amplification curves of ezrin, radixin, moesin, and ABCB1 together with that of beta-actin (internal control) in LS180 and Caco-2 cells as determined by real-time quantitative reverse transcription-polymerase chain reaction. (C) Confocal laser scanning microscopy analysis of ERM and P-gp to visualize the subcellular distribution in LS180 cells. In a three-dimensional reconstruction of optically sectioned LS180 cells, ezrin, radixin, and moesin (red) were detected on the plasma membrane and preferentially colocalized with P-gp (green) on the plasma membrane. Scale bars: 20 mum. All data are representative of at least three independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 10.1371/journal.pone.0250889.g005 Fig 5 Effect of siRNAs targeting ezrin, radixin, or moesin on the ABCB1 mRNA and P-gp expression on the surface of LS180 cells under the acidic condition. Cells were treated with the transfection medium (Untreated), transfection reagent (Lipofectamine), nontargeting control (NC) siRNA, and specific siRNAs for ABCB1, ezrin, radixin, or moesin and then incubated for 3 days under the acidic condition with pH 6.5. (A) The expression of ABCB1 mRNA in cells treated with each siRNA relative to that in cells treated with the transfection reagent alone was determined by real-time quantitative reverse transcription-polymerase chain reaction. n = 3, *** p < 0.001 vs. Lipofectamine. (B) An overlay of the representative histograms for the expression peak of Alexa Fluor 488-labeled P-gp on the surface of the plasma membrane of LS180 cells treated with the transfection medium (Untreated; black line), transfection reagent (Lipofectamine; gray line), ABCB1 siRNA (purple line), and ezrin siRNA (pink line), as measured by flow cytometry. The calculated relative mean fluorescence intensity peaks of P-gp on the surface of the plasma membrane for all the treatments are shown in (C). n = 4, ** p < 0.01, * p < 0.05 vs. Lipofectamine. All data are expressed as the mean +- SEM and were analyzed by a one-way ANOVA followed by Tukey's test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 P-gp targeting comparison between LightSpot (r) -FL-1 staining and anti-P-gp immunostaining on DU4475 and SUM1315 TNBC cell lines. Co-staining was carried out, combining LightSpot (r) -FL-1 with anti-P-gp immunostaining. Pictures of LightSpot (r) -FL-1 labeling (green GFP signal) are presented for DU4475 ( a ) and SUM1315 cells ( c ). Similarly, immunostaining pictures (red Cy5 signal) are presented for DU4475 ( b ) and SUM1315 cells ( d ). Merged pictures are presented for DU4475 ( e ) and SUM1315 ( g ) cells. A line profile was drawn on merged pictures, superimposing both LightSpot (r) -FL-1 and anti-Pgp immunostaining signals. The fluorescence intensity spectra across the line profile are shown with the line profile graphic on DU4475 cells ( f ) and SUM135 cells ( h ). All pictures were acquired with the Cytation(tm)3 MV (BioTek (r) , M = 4 X, scale bar = 25 um).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 P-gp level quantification comparison after co-staining with LightSpot (r) -FL-1 staining and anti-P-gp immunostaining on several triple-negative breast cancer cell lines. Co-staining was carried out by combining LightSpot (r) -FL-1 staining with anti-P-gp immunostaining on five different TNBC cell lines i.e., DU4475, SW527, HCC1937, MDA-MB-231 and SUM1315. For each cell line, ( a ) both LightSpot-FL staining (green bars) and anti-P-gp immunostaining (red bars) fluorescence intensities were measured using the Gen5 software (BioTek (r) ). Correlation between both stainings was evaluated ( b ) with the correlation line and the R 2 coefficient of determination.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Comparison between anti-P-gp immunostaining and LightSpot (r) -FL-1 fluorescent probe staining for P-gp level expression measure on the SUM1315 cell line in monolayer culture condition after increased Olaparib concentration exposure. SUM1315 cells were treated with 10, 50 or 100 uM Olaparib for 3 h. Cells were then fixed and immunostained, combining anti-P-gp mouse antibody (F4 clone) and a goat anti-mouse secondary antibody, coupled to Alexa Fluor(tm) 568 (red signal) for each dose ( a - d ), and the signal was quantified using Gen5 software (BioTek (r) ) ( e ). LightSpot (r) -FL-1 staining (green signal) was also utilized for the same experimental conditions ( f - i ), and quantified using Gen5 software ( j ). All pictures were acquired with the Cytation(tm)3 MV (BioTek (r) , M = 4 X, scale bar = 50 um). Significances between conditions were indicated as ns (not significant; p > 0.05), and **** p < 0.0001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Development of LightSpot (r) -FL-1 staining on SUM1315 spheroids. SUM1315 spheroids (about 200 um diameter) were incubated with LightSpot (r) -FL-1 at 1 uM and washed several times in D-PBS. Before and between each wash of 30 min, pictures of spheroids were made ( a ) for no wash, ( b ) for 1 wash, ( c ) for 2 washes, ( d ) for 3 washes, and ( e ) for 4 washes. For each wash number condition, spheroid global fluorescent intensity was quantified using Gen5 software (BioTek (r) ) ( f ). Otherwise, SUM1315 spheroids were exposed with increasing Olaparib concentrations for 3 h ( g ) or for 6 h ( h ). After the LightSpot (r) -FL-1 staining, pictures were made for each dose for 3 ( g ) and 6 h ( h ) of Olaparib exposure. For each condition, spheroid global LightSpot (r) -FL-1 fluorescent intensity was quantified for 3 h of Olaparib-exposed spheroids ( g ) and for 6 h Olaparib-exposed spheroids ( h ). All pictures were acquired with the Cytation(tm)3 MV (BioTek (r) , M = 4 X, scale bar = 50 um). Significances between conditions were indicated as ns (not significant; p > 0.05), ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Effect of Nedd4 knockdown on Abeta export from CHO-APP cells. CHO-APP cells were transfected with Nedd4 or P-gp siRNA or universal control. Following the initial 24 h transfection period, transfection media was changed to fresh full culture media. After a further 24 h incubation period, cell media were collected for quantification of Abeta 40 content by ELISA, and cells were harvested for Western blot analysis. The column graph shows the relative secretion of Abeta 40 peptides into media over 24 h compared with control. Immunoblots confirm the knockdown of Nedd4 and enhancement of P-gp protein expression in Nedd4-siRNA treated cells compared with control. Tubulin was used as a loading control. Data reflect the mean +- SEM of three independent experiments. ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Duodenum Intestine-Chip shows the presence of major intestinal drug transporters and correct localization and function of efflux pump MDR1 (P-gp). ( a ) Comparison of the relative average gene expression levels of drug efflux (MDR1, BCRP, MRP2, MRP3) and uptake (PEPT1, OATP2B1, OCT1, SLC40A1) transporters in Caco-2 Intestine-Chip, Duodenum Intestine-Chip, both assessed on day 8 of culture, and RNA isolated directly from the duodenal tissue (Duodenum). The results show that Duodenum Intestine-Chip expresses drug transport proteins at the levels close to human duodenal tissue. Note, the expression of OATP2B1 and OCT1 in Caco-2 Intestine-Chip were significantly higher than in human duodenum while the difference between Duodenum Intestine-Chip and adult duodenum is not significant. Each value represents average gene expression +- s.e.m (error bars) from three independent experiments, each involving Duodenum Intestine-Chip established from a tissue of three different donors (three chips/donor), RNA tissue from three independent biological specimens, and Caco-2 Intestine-Chip (three chips). Values are shown relative to the duodenal tissue expressed as 1, two-way ANOVA, ****p

MA5-13854

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