Anti-STAT4 (phospho Y693) antibody [EPR27081-49]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (AB313630)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) treated with 50ng/ml IL-12 for 5h (Red) / Untreated No-GFP-CD16.NK-92 (Green) cells labelling STAT4 (phospho Y693) with ab313630 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (AB313630)

STAT4 (phospho Y693) was immunoprecipitated from 0.35 mg No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) treated with 50 ng/mL IL-12 for 5 hours whole cell lysate with ab313630 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab313630 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) treated with 50 ng/mL IL-12 for 5 hours whole cell lysate Lane 2 : ab313630 IP in No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) treated with 50 ng/mL IL-12 for 5 hours whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab313630 in No-GFP-CD16.NK-92 treated with 50 ng/mL IL-12 for 5 hours whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (ab313630) at 1/30 dilution

All lanes:

No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) treated with 50 ng/mL IL-12 for 5 hours whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (AB313630)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The antibody detects Human STAT4 (phospho Y693).

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-STAT4 antibody (ab284408) staining at 1/1000 dilution.

In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (ab313630) at 1/1000 dilution

Lane 1:

293T cells transfected with a Human STAT4 expression vector containing a His-tag were treated with treated with 100 mM Calyculin A for 30 minutes whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a Human STAT4 (Y693F mutation) expression vector containing a His-tag were treated with 100 mM Calyculin A for 30 minutes 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 1/100000 dilution

Observed band size: 85 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (AB313630)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-STAT4 antibody (ab284408) staining at 1/1000 dilution.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (ab313630) at 1/1000 dilution

Lane 1:

Untreated No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

No-GFP-CD16.NK-92 treated with /mL IL-12 for 5 hours whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated No-GFP-CD16.NK-92 whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 4:

No-GFP-CD16.NK-92 treated with /mL IL-12 for 5 hours whole cell lysate (phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 85 kDa

true

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-STAT4 (phospho Y693) antibody [EPR27081-49] (AB313630)

Dot blot analysis of STAT4 (phospho Y693) using ab313630 at 1 : 1000 (0.476 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Lane 1 : STAT4 (phospho Y693) peptide a Lane 2 : STAT4 (phospho Y693) peptide b Lane 3 : STAT4 non-phospho peptide c Exposure time : 180 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST