Phospho-STAT3 (S727) Recombinant Rabbit Monoclonal Antibody [SY24-09]

☑ Cell treatment (CT)

Western blot analysis of Phospho-STAT3(S727) on NIH/3T3 cell lysates.

Lane 1: NIH/3T3 cells, whole cell lysates, 10 μg/lane.
Lane 2: NIH/3T3 cells were starved 6h, whole cell lysates, 10 μg/lane.
Lane 3/4: NIH/3T3 cells were starved 6h, then treated with 200 ng/ml EGF for 10 min, whole cell lysates, 10 μg/lane.
Lane 5: NIH/3T3 cells were starved 6h, then treated with 200 ng/ml EGF for 10 min, and then treated with 2.8μg/ul lambda-PP for 30 minutes, whole cell lysates, 10 μg/lane.

Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody Anti-Phospho-STAT3(S727)(ET1607-39, 1/500) , Anti-STAT3 antibody ( ET1607-38, 1/500) and Anti-GAPDH antibody (ET1601-4, 1/10,000)was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1/200,000 dilution was used for 1 hour at room temperature.

Predicted band size: 88 kDa
Observed band size: 88 kDa
Exposure time: Lane 1/2/3 1 minute 28 seconds
Lane 4/5 30 seconds

☑ Knockdown (KD)

Western blot analysis of Phospho-STAT3(S727) with anti-Phospho-STAT3(S727) antibody [SY24-09] (ET1607-39) at 1/1,000 dilution.

Lane 1: Wild-type CT26.wt whole cell lysate.
Lane 2: STAT3 knockdown CT26.wt whole cell lysate.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-Phospho-STAT3(S727) antibody (ET1607-39, 1/1,000) and Anti-HSP90 antibody (ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1/200,000 dilution was used for 1 hour at room temperature.

Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

Western blot analysis of Phospho-STAT3(S727) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1607-39, 1/1,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Positive control:
Lane 1: Hela cell lysate
Lane 2: A431 cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: Jurkat cell lysate

☑ Cell treatment (CT)

Western blot analysis of Phospho-STAT3 (S727) on different lysates with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/1,000 dilution.

Lane 1: PC-12 cell lysate (20 µg/Lane)
Lane 2: C6 cell lysate (20 µg/Lane)
Lane 3: C6 treated with 200ng/mL TPA for 35 minutes cell lysate (20 µg/Lane)
Lane 4: Rat cerebellum tissue lysate (40 µg/Lane)

Predicted band size: 88 kDa
Observed band size: 88 kDa

Exposure time: 1 minute 50 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1607-39) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

ICC staining of Phospho-STAT3 (S727) in Hela cells (green).

Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-39, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

ICC staining of Phospho-STAT3 (S727) in NIH/3T3 cells (green).

Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1607-39, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

Immunocytochemistry analysis of Huh7 cells labeling Phospho-STAT3 (S727) with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1:500 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-39) at 1:500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1:500 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-39) at 1:500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1:500 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-39) at 1:500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-39) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Rabbit anti-Phospho-STAT3 (S727) antibody (ET1607-39) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-39) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.