All lanes : Anti-STAT6 (phospho Y641) antibody [EPR22599-78] (ab263947) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) serum starved for 24 hours, whole cell lysate at 10 µg
Lane 2 : RAW264.7 serum starved for 24 hours, then treated with 100 ng/ml mIL-4 for 15 minutes, whole cell lysate 10 ug

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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

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

Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment (PMID: 21411736,15044251) The molecular weight observed is consistent with what has been described in the literature (PMID: 24708771).

STAT6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13. 

Exposure time: 15 seconds

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling STAT6 (phospho Y641) with ab263947 at 1:5000 dilution (0.106 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human kidney (panel A), no staining after alkaline phosphatase treatment (panel B. PMID: 8085155, 16181056). The section was incubated with ab263947 for 15 mins at RT. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min (Red) / Untreated control (Green) cells labelling STAT6 (phospho Y641) with ab263947 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

STAT6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13.

All lanes : Anti-STAT6 (phospho Y641) antibody [EPR22599-78] (ab263947) at 1/5000 dilution

Lane 1 : 2.4G2 (rat B cell lymphoma B lymphocyte) serum starved for 24 hours, whole cell lysate at 10 µg
Lane 2 : 2.4G2 serum starved for 24 hours, then treated with 100 ng/ml rIL-4 for 15 minutes, whole cell lysate 10 ug

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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

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

Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment (PMID: 21411736,15044251) The molecular weight observed is consistent with what has been described in the literature (PMID: 24708771).

STAT6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13.

Exposure time: 37 seconds

All lanes : Anti-STAT6 (phospho Y641) antibody [EPR22599-78] (ab263947) at 1/5000 dilution

Lane 1 : Daudi (human Burkitts lymphoma lymphoblast) serum starved for 24 hours, whole cell lysate at 10 µg
Lane 2 : Daudi serum starved for 24 hours, then treated with 100 ng/ml IL-4 for 15 minutes, whole cell lysate 10 ug

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 110,75,81 kDa why is the actual band size different from the predicted?

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

Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment (PMID: 21411736,15044251) This antibody recognizes three isoforms of STAT6, isoform 1 (110 kDa), isoform 2 (75 kDa), isoform 3 (81 kDa), which is consistent with the Uniprot annotation.
STAT6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13.

Exposure time: 15 seconds

Dot blot analysis of STAT6 (phospho Y641) peptide (Lane 1 STAT6 non-phospho peptide peptide (Lane 2), labelling STAT 6 (pY641) with purified ab263947 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 26 seconds.

STAT6 (phospho Y641) was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate 10ug with ab263947 at 1:30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263947 at 1:1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:5000 dilution.

Lane 1: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate 10ug

Lane 2: ab263947 IP in RAW 264.7 was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab263947 in RAW 264.7 was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

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

Exposure time: 2 min

STAT6 (phospho Y641) was immunoprecipitated from 0.35 mg Daudi (Human Burkitt's lymphoma lymphoblast) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate 10ug with ab263947 at 1:30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263947 at 1:1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:5000 dilution.

Lane 1: Daudi (Human Burkitt's lymphoma lymphoblast) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate 10ug

Lane 2: ab263947 IP in Daudi was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab263947 in Daudi was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

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

STAT6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13.

Exposure time: 1 min

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Daudi (Human Burkitt's lymphoma lymphoblast) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min (Red)/Untreated control (Green) cells labelling STAT6 (phospho Y641) with ab263947 at 1/500 dilution(Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

STAT6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13.

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling STAT6 (phospho Y641) with ab263947 at 1:5000 dilution (0.106 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat liver (panel A), no staining after alkaline phosphatase treatment (panel B). The section was incubated with ab263947 for 15 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling STAT6 (phospho Y641) with ab263947 at 1/5000 dilution (0.106 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse kidney (panel A), no staining after alkaline phosphatase treatment (panel B, PMID: 23155424). The section was incubated with ab263947 for 15 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.