Anti-STAT5a antibody [E289]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5a antibody [E289] (AB32043)

Tissue Microarrays stained for "Anti-STAT5a antibody [E289]" using "ab32043"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab32043 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5a antibody [E289] (AB32043)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast tissue sections labeling STAT5a with purified ab32043 at 1 : 1000 dilution (0.12 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use). PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-STAT5a antibody [E289] (AB32043)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling STAT5a with purified ab32043 at 1/100 dilution (1 μg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluorr^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT5a antibody [E289] (AB32043)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (human T cell leukemia T lymphocyte) cells labeling STAT5a with purified ab32043 at 1 : 100 (1.2 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5a antibody [E289] (AB32043)

Unpurified ab32043 at 1/250 dilution, staining human squamous lung carcinoma by Immunohistochemistry, paraffin-embedded tissue. Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using (Tris/EDTA buffer, pH 9.0).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT5a antibody [E289] (AB32043)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells labelling STAT5a with unpurified ab32043 at 1/500. 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.
Control : PBS only.
Nuclear counter stain : DAPI.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-STAT5a antibody [E289] (AB32043)

Overlay histogram showing Jurkat cells stained with unpurified ab32043 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32043, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IP

Unknown

Immunoprecipitation - Anti-STAT5a antibody [E289] (AB32043)

ab32043 (purified) at 1 : 20 dilution (0.6ug) immunoprecipitating in TF-1 whole cell lysate. TF-1 (Human Erythroleukemia erythroblast) whole cell lysate 10ug
Lane 2 (+) : ab32043 & TF-1 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32043 in TF-1 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-STAT5a antibody [E289] (ab32043)

Predicted band size: 90 kDa

Observed band size: 92 kDa

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• WB

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Western blot - Anti-STAT5a antibody [E289] (AB32043)

All lanes:

Western blot - Anti-STAT5a antibody [E289] (ab32043) at 1/1000 dilution

All lanes:

A431 cell lysate

Predicted band size: 90 kDa

Observed band size: 92 kDa

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• WB

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Western blot - Anti-STAT5a antibody [E289] (AB32043)

All lanes:

Western blot - Anti-STAT5a antibody [E289] (ab32043) at 1/1000 dilution

All lanes:

Western blot - Recombinant Human STAT5a protein (<a href='/en-us/products/proteins-peptides/recombinant-human-stat5a-protein-ab84627'>ab84627</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 90 kDa

true

Exposure time: 20s

• WB

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Western blot - Anti-STAT5a antibody [E289] (AB32043)

All lanes:

Western blot - Anti-STAT5a antibody [E289] (ab32043) at 1/1000 dilution

Lane 1:

Mouse brain lysates at 15 µg

Lane 2:

Rat brain lysates at 15 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: 90 kDa

Observed band size: 92 kDa

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• WB

Unknown

Western blot - Anti-STAT5a antibody [E289] (AB32043)

All lanes:

Western blot - Anti-STAT5a antibody [E289] (ab32043) at 1/20000 dilution

All lanes:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates 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

Predicted band size: 90 kDa

Observed band size: 92 kDa

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• WB

CiteAb

Western blot - Anti-STAT5a antibody [E289] (AB32043)

STAT5a western blot using anti-STAT5a antibody [E289] ab32043. Publication image and figure legend from Wang, W., Liu, Y., et al., 2018, Oncogenesis, PubMed 30563983.

ab32043 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32043 please see the product overview.

miR-100 increases IL-1ra secretion through stat5a-mediated transcriptional regulation.a Supernatants of RAW264.7 cells transfected with miR-100 mimic and nc mimic were collected and secreted cytokines were measured by the mouse cytokine array panel. b IL-1ra mRNA expression was measured by qPCR in miR-100 mimic transfected RAW264.7 cells. c IL-1ra+ cells percentage in PMs transfected with miR-100 mimic were measured by flow cytometry. d IL-1ra expression in miR-100 antagomir-treated 4T1 mouse breast cancer tissues was detected by ELISA. e, f qPCR and Western blot to detect IL-1ra expression in RAW264.7 cells transfected with siRNAs targeting several candidate TFs. g Western blot to detect Stat5a and Stat5b expression in RAW264.7 cells transfected with miR-100 mimic or treated with rapamycin. h Stat5a binding sites were predicted by ALLGEN database in IL-1ra promoter region from -3k to +1k. i Three stat5a binding sites were validated by qPCR in genomic DNA extracted from RAW264.7 cells. Data were shown as mean ± s.e.m. from three independent experiments; **p < 0.01, *p < 0.05

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