Phospho-IRE1 (S724) Recombinant Rabbit Monoclonal Antibody [PSH03-35]

☑ Cell treatment (CT)

Western blot analysis of Phospho-IRE1 (S724) on different lysates with Rabbit anti-Phospho-IRE1 (S724) antibody (HA721980) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)
Lane 3: Jurkat cell lysate (20 µg/Lane)
Lane 4: Jurkat treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)

Predicted band size: 110 kDa
Observed band size: 110 kDa

Exposure time: 5 minutes 10 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 (HA721980) 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.

Western blot analysis of Phospho-IRE1 (S724) on different lysates with Rabbit anti-Phospho-IRE1 (S724) antibody (HA721980) at 1/1,000 dilution.

Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
Lane 2: NIH/3T3 treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)
Lane 3: C2C12 cell lysate (20 µg/Lane)
Lane 4: C2C12 starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)

Predicted band size: 110 kDa
Observed band size: 110 kDa

Exposure time: 24 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 (HA721980) 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.

☑ Cell treatment (CT)

Immunocytochemistry analysis of Jurkat cells treated with 100nM Calyculin A for 30 minutes labeling Phospho-IRE1 (S724) with Rabbit anti-Phospho-IRE1 (S724) antibody (HA721980) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-IRE1 (S724) antibody (HA721980) at 1/500 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 (M1305-2, 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.

☑ Cell treatment (CT)

Immunohistochemical analysis of paraffin-embedded human pancreas tissue untreated / treated with λpp with Rabbit anti-Phospho-IRE1 (S724) antibody (HA721980) 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 (HA721980) 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.

☑ Cell treatment (CT)

Flow cytometric analysis of HeLa cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-IRE1 (S724).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721980, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).