Anti-IRE1 (phospho S724) antibody

• WB

Supplier Data

Western blot - Anti-IRE1 (phospho S724) antibody (AB48187)

HeLa cells were treated (+) or untreated (-) with 10 mM DTT for 60 min to activate the UPR. Total protein was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% BSA in TBST.

All lanes:

Western blot - Anti-IRE1 (phospho S724) antibody (ab48187) at 2 µg/mL

Lane 1:

Untreated HeLa cells (Batch 1 ab48187)

Lane 2:

DTT-treated HeLa cells (Batch 1 ab48187)

Lane 3:

Untreated HeLa cells (Batch 2 ab48187)

Lane 4:

DTT-treated HeLa cells (Batch 2 ab48187)

Secondary

All lanes:

Anti-Rabbit IgG HRP

Predicted band size: 110 kDa

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• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRE1 (phospho S724) antibody (AB48187)

Paraffin-embedded human spleen tissue stained for IRE1 using ab48187 at 1/300 dilution for 1 hour at room temperature in immunohistochemical analysis. DAB staining. Counter stained with hematoxylin.

• WB

Lab

Western blot - Anti-IRE1 (phospho S724) antibody (AB48187)

The blots were produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membranes were blocked for an hour. Membrane 2 was incubated with alkaline phosphatase (AP; 100 U per mL) for one hour, whilst membrane 1 was treated with AP-buffer only, before being incubated with ab48187 (rabbit anti-IRE1 antibody diluted 1 : 2000) and loading control ab125247 (mouse anti-GAPDH antibody; diluted 1 : 10,000) for 24 hours at 4°C. Antibody binding was detected using infrared (IR)-labelled goat anti-rabbit (green) antibody and IR-labelled goat anti-mouse (red) at 1 : 10,000 dilutions for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IRE1 (phospho S724) antibody (ab48187) at 1/2000 dilution

Lane 1:

HeLa cells 30 nM Calyculin A: AP-buffer at 20 µg

Lane 2:

HeLa cells 30 nM Calyculin A: Alkaline Phosphatase at 20 µg

Secondary

All lanes:

infrared (IR)-labelled goat anti-rabbit (green) antibody and IR-labelled goat anti-mouse (red) at 1/10000 dilution

Predicted band size: 110 kDa

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• WB

Unknown

Western blot - Anti-IRE1 (phospho S724) antibody (AB48187)

All lanes:

Western blot - Anti-IRE1 (phospho S724) antibody (ab48187) at 1/1000 dilution

Lane 1:

Min6 cells untreated

Lane 2:

Min6 cells treated with glucose for 3 hours at 5 mM

Lane 3:

Min6 cells treated with glucose for 3 hours at 20 mM

Predicted band size: 110 kDa

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• ELISA

Lab

ELISA - Anti-IRE1 (phospho S724) antibody (AB48187)

Serially diluted ab48187 was bound to immobilised Phospho peptide (133861) - or Control peptide (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.

• WB

Unknown

Western blot - Anti-IRE1 (phospho S724) antibody (AB48187)

All lanes:

Western blot - Anti-IRE1 (phospho S724) antibody (ab48187) at 1/2000 dilution

Lane 1:

Cell lysate prepared from COS-7 Untransfected cells

Lane 2:

Cell lysate prepared from COS-7 cells expressing wild type IRE1 alpha

Lane 3:

Cell lysate prepared from COS-7 cells expressing kinase-dead IRE1 alpha

Predicted band size: 110 kDa

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• WB

CiteAb

Western blot - Anti-IRE1 (phospho S724) antibody (AB48187)

IRE1 western blot using Anti-IRE1 (phospho S724) antibody, ab48187. Publication image and figure legend from Navarro-Betancourt, J. R., Papillon, J., et al.,2020, Cell Death Discov, PubMed 33298866.

ab48187 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab48187 please see the product overview.

IRE1α signaling is adaptive in cultured GECs subjected to proteotoxic stress.A, B Incubation with TM for 24 h stimulated the upregulation of total IRE1α, robust XBP1 splicing, and mild apoptosis (caspase-3 cleavage) in control (Ctrl) GECs. Quantification of IRE1α phosphorylation was normalized to the actin signal. IRE1α phosphorylation was absent in IRE1α KO GECs but was not affected by 4μ8C, compared to control GECs. IRE1α KO and 4μ8C-treated GECs show undetectable XBP1 splicing and greater apoptosis (immunoblots). C–F TM increased the expression of ER chaperones in control GECs. C, D Upregulation of GRP94, BiP and MANF was impaired in IRE1α KO GECs, and these results were generally recapitulated by 4μ8C. E, F The chaperone ERdj3 is a glycosylated protein. TM inhibits glycosylation and induces an underglycosylated band of ~34 kDa (both bands were used for quantification). Upregulation of ERp57 and ERdj3 was not affected by IRE1α inhibition. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Three experiments performed in duplicate.

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