Anti-IRS1+IRS2 antibody [EP263Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling IRS1 with purified ab40777 at a dilution of 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077 an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291 a mouse anti-tubulin (1/1000) and ab150120 an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/250) and secondary antibody ab150120 an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody ab150077 an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma labelling IRS1 with unpurified ab40777.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling IRS1 with purified ab40777 at a dilution of 1/100. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051 a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Intracellular Flow Cytometry analysis of MCF-7 (human breast carcinoma) cells labeling IRS1 with unpurified ab40777 at 1/90 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• WB

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Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

All lanes:

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (ab40777) at 1/1000 dilution

All lanes:

HeLa cell lysate at 10 µg

Observed band size: 170 kDa

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• WB

Lab

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (ab40777) at 1/10000 dilution

All lanes:

HEK293 whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 170 kDa

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• WB

Lab

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (ab40777)

All lanes:

HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 52 kDa

Observed band size: 170 kDa

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• WB

Supplier Data

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Blocking and diluting buffer and concentration : 5% NFDM /TBST. Exposure time : Lane 1, 2, 4, 5 , 6 : 1 second. Lane 3 : 80 seconds.

Lanes 1 - 3:

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (ab40777) at 1/1000 dilution

Lanes 4 - 6:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (<a href='/en-us/products/primary-antibodies/ddddk-tag-binds-to-flag-tag-sequence-antibody-epr20018-251-ab205606'>ab205606</a>) at 1/1000 dilution

Lanes 1 and 4:

HEK-293T transfected with an empty vector whole cell lysate at 20 µg

Lanes 2 and 5:

HEK-293T transfected with a human IRS1 expression vector containing a Myc/DDK tag whole cell lysate at 20 µg

Lanes 3 and 6:

HEK-293T transfected with a human IRS2 expression vector containing a Myc/DDK tag whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 170 kDa

false

• WB

CiteAb

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (AB40777)

Western Blotting using Anti-IRS1+IRS2 antibody [EP263Y], ab40777. Publication image from Eyler, C. E. et al., 2020, Genome Biol, 32669109. Legend direct from paper.

Analysis of clonal cultures identifies distinct but functionally parallel chromosomal amplifications in “jackpot” drug resistant lineages. a Schematic depicts isolation of single cells and derivation of clonal cultures from multiple dasatinib-treated replicates of Experiments #1 (day 70; left) and 2 (day 56; right). b Chart details characteristics of those clonal cultures isolated from dasatinib experiments that were analyzed by low-coverage whole-genome sequencing (WGS), compared to drug-naïve GSCs (top row). Clones that exhibited features consistent with induced drug persistence (epigenetic) in these experiments are shown in the top part of chart. Clones that exhibited features consistent with genetic (acquired or preexisting) resistance are shown in the bottom part of chart. For each single-cell-derived clone, the chart indicates the experiment and replicate from which it was derived (Clone ID), the DNA barcode identity (Lineage ID), and the rank and abundance of the respective lineage in the gDNA analysis of the corresponding experiment (day 70 for Experiment #1; day 56 for Experiment #2). Genomic tracks depict read density in low-coverage WGS over chromosomal bands 13q34 (containing IRS2) and 2q36 (containing IRS1). The final column summarizes the extent to which the clone loses dasatinib resistance after a drug-free washout period. c Single-cell-derived clones were subjected to a drug-free washout period and then re-challenged with dasatinib. Plot depicts growth in the presence of dasatinib, relative to drug-naïve GSC8 cells (Y-axis); all conditions were treated independently in at least 4 replicates. Circles represent the mean relative growth of individual clonal cultures. Horizontal black bars reflect the mean growth of clones from the indicated lineage (top). d Barplots depict relative growth of drug-naïve GSC8 cells transduced with IRS1, IRS2, or luciferase expression constructs (**, p < 0.01 by two-tailed Student’s t test; standard error bars depicted). Cells were grown at the indicated dasatinib concentrations. Western blot shows IRS1, IRS2, and Actin protein expression in the indicated GSC8 cultures. Overexpression of IRS1 or IRS2 confers dasatinib resistance

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