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

Usd: 385

Specification

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Catalog# HA721980

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

Application

  • WB

  • IF-Cell

  • IHC-P

  • FC

Reactivity

  • Human

  • Mouse

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

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

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser724 of human IRE1.

Species Reactivity

Human, Mouse

Validated Applications

WB, IF-Cell, IHC-P, FC

Target Molecular Weight

Predicted band size: 110 kDa

Positive Control

HeLa starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate, Jurkat treated with 100nM Calyculin A for 30 minutes cell lysate, NIH/3T3 treated with 100nM Calyculin A for 30 minutes cell lysate, C2C12 starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate, Jurkat cells treated with 100nM Calyculin A for 30 minutes, human pancreas tissue, HeLa cells treated with 100nM Calyculin A for 30 minutes.

Conjugation

unconjugated

Clone Number

PSH03-35

RRID

AB_3096844

Product Features

Form

Liquid

Concentration

1 mg/mL.(The concentration of this product may be batch-dependent)

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000

  • IF-Cell

  • 1:500

  • IHC-P

  • 1:500

  • FC

  • 1:1,000

Target

Function

The serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 α (IRE1α) is an enzyme that in humans is encoded by the ERN1 gene. The protein encoded by this gene is the ER to nucleus signalling 1 protein, a human homologue of the yeast Ire1 gene product. This protein possesses intrinsic kinase activity and an endoribonuclease activity and it is important in altering gene expression as a response to endoplasmic reticulum-based stress signals (mainly the unfolded protein response). Two alternatively spliced transcript variants encoding different isoforms have been found for this gene.

Background References

1. Huang R et al. IRE1 signaling regulates chondrocyte apoptosis and death fate in the osteoarthritis. J Cell Physiol. 2022 Jan

2. Grandjean JMD et al. Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming. Nat Chem Biol. 2020 Oct

Subcellular Location

Endoplasmic reticulum membrane; nucleus.

UNIPROT

SwissProt: O75460 Human

SwissProt: Q9EQY0 Mouse

Synonyms

Endoplasmic reticulum (ER) to nucleus signalling 1 antibody

Endoplasmic reticulum to nucleus signaling 1 antibody

Endoplasmic reticulum-to-nucleus signaling 1 antibody

Endoribonuclease antibody

ER to nucleus signaling 1 antibody

ERN 1 antibody

Ern1 antibody

ERN1_HUMAN antibody

hIRE 1p antibody

hIRE1p antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-IRE1 (S724) on different lysates with Rabbit anti-Phospho-IRE1 (S724) antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate (20 µg/Lane)<br />Lane 2: HeLa starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)<br />Lane 3: Jurkat cell lysate (20 µg/Lane)<br />Lane 4: Jurkat treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)<br /><br />Predicted band size: 110 kDa<br />Observed band size: 110 kDa<br /><br />Exposure time: 5 minutes 10 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ 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 (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/1,000 dilution.<br /><br />Lane 1: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 2: NIH/3T3 treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)<br />Lane 3: C2C12 cell lysate (20 µg/Lane)<br />Lane 4: C2C12 starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)<br /><br />Predicted band size: 110 kDa<br />Observed band size: 110 kDa<br /><br />Exposure time: 24 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunocytochemistry analysis of Jurkat cells treated with 100nM Calyculin A for 30 minutes labeling Phospho-IRE1 (S724) with Rabbit anti-Phospho-IRE1 (S724) antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/500 dilution.<br /><br />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 (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) 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.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    ☑ 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunohistochemical analysis of paraffin-embedded human pancreas tissue untreated / treated with λpp with Rabbit anti-Phospho-IRE1 (S724) antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) at 1/500 dilution.<br /><br />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 ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>) 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)

    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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Flow cytometric analysis of HeLa cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-IRE1 (S724).<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/HA721980" style="font-weight: bold;text-decoration: underline;">HA721980</a>, 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&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    ☑ 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).

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Citation

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