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<strong>Figure 1 Western Blot Validation in Mouse A20 Cell Lysate</strong><br>Loading: 15 μg of lysates per lane.Antibodies: IRE1p 3655 (A: 0.5 μg/mL, B: 1 μg/mL, C: 2 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 KO Validation in HeLa Cells</strong><br>Loading: 10 μg of WT cell lysates (lane 1) or IRE1P KO cell lysates (lane 2).Antibodies: IRE1P 3655 (0.5 μg/mL) and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Western Blot Validation in Human Cell Lines</strong><br>Loading: 15 μg of lysates per lane.Antibodies: IRE1p 3655 (0.4 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.Lane 1: Caco-2, Lane2: SK-N-SH
<strong>Figure 4 Western Blot Validation in Rat Brain Tissue Lysate</strong><br>Loading: 15 μg of lysates per lane.Antibodies: IRE1p 3655 (A: 0.5 μg/mL, B: 1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunofluorescence Validation of IRE1p in Mouse A20 Cells</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed A20 Cells labeling IRE1P with 3655 at 2 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
<strong>Figure 6 Immunocytochemistry Validation of IRE1p in Mouse A20 Cells</strong><br>Immunocytochemical analysis of A20 cells using anti-IRE1p antibody (3655) at 1 μg/ml. Cells was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 7 Immunofluorescence Validation of IRE1p in Human Small Intestine Tissue</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed Human Small Intestine Tissue labeling IRE1p with 3655 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
<strong>Figure 8 Immunohistochemistry Validation of IRE1p in Human Small Intestine Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded Human Small Intestine Tissue using anti-IRE1P antibody (3655) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 9 Immunofluorescence Validation of IRE1p in Rat Small Intestine Tissue</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed Rat Small Intestine Tissue labeling IRE1p with 3655 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
<strong>Figure 10 Immunohistochemistry Validation of IRE1p in Rat Small Intestine Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded Rat Small Intestine Tissue using anti-IRE1P antibody (3655) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 11 Induced Expression Validation of IRE1p in human umbilical vein endothelial cells (HUVECs) (Wang et al., 2019) </strong><br>IRE1p expression was examined by Western blot analysis with anti-IRE1p antibodies (3655). IRE1p was increased in HUVEC cells treated with 10 μM 20(S)PPD for 6 to 8 hours compared with control cells.
Flow Cytometry Validation of IRE1p in Jurkat Cells 1X10^6/mL Jurkat cells were intracellularly stained with 2 μg/mL of IRE1p antibody (red) or Rabbit IgG isotype control (black) at RT for 30 min, followed by PSFlo 488 conjugated goat anti-rabbit IgG secondary antibody staining (1:1000) at RT for 30 min. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100 in PBS.
Figure 1 Western Blot Validation in Mouse A20 Cell Lysate
Loading: 15 μg of lysates per lane.Antibodies: IRE1p 3655 (A: 0.5 μg/mL, B: 1 μg/mL, C: 2 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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IRE1p Antibody

CATALOG NUMBER: 3655

Clonality:
Polyclonal
Tested Applications:
ELISA, FC, ICC, IF, IHC-P, WB
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Immunogen:
Anti-IRE1p antibody (3655) was raised against a peptide corresponding to 16 amino acids near the carboxy terminus of human IRE1P.
The immunogen is located within the last 50 amino acids of IRE1p.
Conjugate:
Unconjugated
Tested Applications:
ELISA, FC, ICC, IF, IHC-P, WB
Application Note:
WB: 0.5-2 μg/mL; ICC: 1 μg/mL; IF: 2-20 μg/mL; IHC: 2 μg/mL; FC: 2 μg/mL.
Antibody validated: Western Blot in human, mouse and rat samples; Immunocytochemistry in mouse samples; Immunofluorescence in human, mouse and rat samples; Immunohistochemistry in human and rat samples; Flow Cytometry in human samples. All other applications and species not yet tested.
Positive Control 1:
Cat. No. 1288 - A20 Cell Lysate
Positive Control 2:
Cat. No. 17-208 - A-20 Cell Slide
Positive Control 3:
Cat. No. 1463 - Rat Brain Tissue Lysate
Predicted Molecular Weight:
Predicted: 110kD
Observed: 110kD
Validation:

KO validation (Figure 2): Anti-IRE1p antibody (3655) specificity was further verified by IRE1P specific knockout. IRE1p signal was disrupted in IRE1p knockout HeLa cells in comparison with that in control HeLa cells.

Induced Expression Validation (Figure 11): IRE1p expression detected by anit-IRE1p antibodies was up-regulated by 20(S)PPD treatment.

Isoforms:
Human IRE1p has 2 isoforms, including isoform 1 (977aa, 110kD) and isoform 2 (70aa, 6.6kD). Mouse IRE1p also has two isoforms, including isoform 1 (977aa, 110kD) and isoform 2 (408aa, 45kD). Rat IRE1p has one isoform (965aa, 109kD). 3655 can detect human, mouse and rat.
Purification:
IRE1p Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
IRE1p Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
IRE1p antibody can be stored at 4°C for three months and -20°C, stable for up to one year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
ERN1
Additional Names:
IRE1p Antibody: IRE1, IRE1P, IRE1a, hIRE1p, IRE1, Endoplasmic reticulum-to-nucleus signaling 1
Protein Accession Number:
O75460
Protein Gi Number:
193806335
Ncbi Gene Id Number:
2081
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
IRE1p Antibody: Accumulation of malfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) and the upregulation of the ER molecular chaperones GRP78 and GRP 94. These proteins are normally bound to ER transmembrane proteins such as IRE1p and ATF6 but ER stress causes their dissociation. This allows IRE1p, a serine-threonine protein kinase to transduce the unfolded protein signal from the ER to the nucleus. IRE1p also has an endoribonuclease activity that is required to splice X-box binding protein (XBP1) mRNA converting it to a potent UPR transcriptional activation. Depletion of IRE1p through the expression of a dominant negative form of IRE1p has no effect on transfected cells, but cell death via apoptosis occurs under stress conditions that cause unfolded proteins to accumulate in the ER. Two alternatively spliced transcript variants encoding different isoforms have been found for this gene.
Background Reference 1:
Little et al. Crit. Rev. Eukaryot. Gene Expr. 1994;4:1-18.
Background Reference 2:
Lee. Methods 2005;35:373-81.
Background Reference 3:
Bertolotti et al. Nat. Cell Biol. 2000;2:326-32.
Background Reference 4:
Shen et al. Dev. Cell 2002;3:99-111.
Citation 1:
Wang et al. 20(S)-protopanaxadiol induces apoptosis in human umbilical vein endothelial cells by activating the PERK-eIF2alpha-ATF4 signaling pathway. J Cell Biochem. 2019;120(4):5085-5096.PMID: 30259568
Citation 2:
Zhang et al. 20(S)-protopanaxadiol activates ER stress to induce cell apoptosis in human umbilical vein endothelial cells. Journal of Shanghai University of Traditional Chinese Medicine 2015;29(3):70-75.PMID:

FOR RESEARCH USE ONLY

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Disclaimer:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.

CATALOG NUMBER:

3655

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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