All lanes : Anti-STAT6 (phospho Y641) antibody [EPR18278-265] (ab188080) at 1/1000 dilution

Lane 1 : Untreated Daudi (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 2 : Daudi whole cell lysate treated with 100 ng/ml interleukin 4 (IL-4) for 15 minutes

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 94 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment according to the literature (PMID: 21411736, PMID: 15044251, PMID: 14660485).

This antibody recognizes three isoforms of STAT6, isoform 1 (110KD), isoform 2 (75KD), isoform 3 (81KD), which is consistent with the Uniprot annotation.

Immunofluorescent analysis of 100% methanol-fixed Daudi (Human Burkitt's lymphoma cell line) cells, treated with interleukin 4 (IL-4) (100 ng/ml) for 15minutes, or untreated, labeling STAT6 (phospho Y641) with ab188080 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The expression increased and there was nuclear translocation of STAT6 after treatment with interleukin 4 (IL-4) (100 ng/ml) for 15minutes (PMID:22226123).

The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

STAT6 (phospho Y641) was immunoprecipitated from 0.35 mg of Daudi (Human Burkitt's lymphoma cell line) whole cell lysate treated with 100 ng/ml IL-4 for 15 minutes with ab188080 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab188080 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1:  Daudi whole cell lysate treated with 100 ng/ml IL-4 for 15 minutes, 10µg (Input). Lane 2: ab188080 IP in Daudi whole cell lysate treated with 100 ng/ml IL-4 for 15 minutes. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab188080 in Daudi whole cell lysate treated with 100 ng/ml IL-4 for 15 minutes.

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

Exposure time: 1 second.

This antibody recognizes three isoforms of STAT6, isoform 1 (110KD), isoform 2 (75KD), isoform 3 (81KD), which is consistent with the Uniprot annotation.

Flow cytometric analysis of 4% paraformaldehyde-fixed Daudi (Human Burkitt's lymphoma cell line) treated/untreated with 100 ng/ml IL-4 for 15 minutes cells labeling STAT6 (phospho Y641) with ab188080 at 1/400 dilution (treated, red; untreated, green) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Dot blot analysis of STAT6 (phospho Y641) labeled with ab188080 at 1/1000 dilution.

Lane 1: STAT6 (phospho Y641) phospho peptide.

Lane 2: STAT6 non-phospho peptide.

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051)  at 1/20000 dilution was used as secondary antibody.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.