Revision 4

#27901Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IF-IC, FC-FP, ChIP, ChIP-seq

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

60

Source/Isotype:

Mouse IgG1

UniProt ID:

#P17861-2

Entrez-Gene Id:

7494

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 2.5 μl of antibody and 10 μg of chromatin (approximately 4 × 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.
Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:100 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:800 - 1:3200
Chromatin IP 1:200
Chromatin IP-seq 1:200

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

XBP-1s (E8C2Z) Mouse mAb recognizes endogenous levels of total XBP-1s protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala285 of human XBP-1s protein.

Background

Following protein synthesis, secretory, intra-organellar, and transmembrane proteins translocate into the endoplasmic reticulum (ER) where they are post-translationally modified and properly folded. The accumulation of unfolded proteins within the ER triggers an adaptive mechanism known as the unfolded protein response (UPR) that counteracts compromised protein folding (1). The transmembrane serine/threonine kinase IRE1, originally identified in Saccharomyces cerevisiae, is a proximal sensor for the UPR that transmits the unfolded protein signal across the ER membrane (2-4). The human homolog IRE1α was later identified and is ubiquitously expressed in human tissues (5). Upon activation of the unfolded protein response, IRE1α splices X-box binding protein 1 (XBP-1) mRNA through an unconventional mechanism using its endoribonuclease activity (6). This reaction converts XBP-1 from an unspliced XBP-1u isoform to the spliced XBP-1s isoform, which is a potent transcriptional activator that induces expression of many UPR responsive genes (6).

  1. Kaufman, R.J. et al. (2002) Nat Rev Mol Cell Biol 3, 411-21.
  2. Nikawa, J. and Yamashita, S. (1992) Mol Microbiol 6, 1441-6.
  3. Cox, J.S. et al. (1993) Cell 73, 1197-206.
  4. Mori, K. et al. (1993) Cell 74, 743-56.
  5. Tirasophon, W. et al. (1998) Genes Dev 12, 1812-24.
  6. Lee, K. et al. (2002) Genes Dev 16, 452-66.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 4
#27901

XBP-1s (E8C2Z) Mouse mAb

Western Blotting Image 1: XBP-1s (E8C2Z) Mouse mAb Expand Image
Western blot analysis of extracts from 293T cells, untreated (-) or treated with tunicamycin #12819 (2 μg/ml, 8 hr; +), using XBP-1s (E8C2Z) Mouse mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 2: XBP-1s (E8C2Z) Mouse mAb Expand Image
Western blot analysis of extracts from SK-N-AS cells, untreated (-) or treated with thapsigargin #12758 (1 μM, 8 hr; +), using XBP-1s (E8C2Z) Mouse mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunofluorescence Image 1: XBP-1s (E8C2Z) Mouse mAb Expand Image
Confocal immunofluorescent analysis of SK-N-AS cells, untreated (left) or treated with thapsigargin #12758 (1 μM, 8 hr; right), using XBP-1s (E8C2Z) Mouse mAb (green) and β-Actin (13E5) Rabbit mAb #4970 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Flow Cytometry Image 1: XBP-1s (E8C2Z) Mouse mAb Expand Image
Flow cytometric analysis of 293T cells, untreated (blue) or treated with tunicamycin #12819 (2 μg/ml, 7 hr; green), using XBP-1s (E8C2Z) Mouse mAb (solid lines) or concentration-matched Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: XBP-1s (E8C2Z) Mouse mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells treated with tunicamycin #12819 (2 μg/ml, 8 hr) and XBP-1s (E8C2Z) Mouse mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across DNAJB9, a known target gene of XBP-1s (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: XBP-1s (E8C2Z) Mouse mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells treated with tunicamycin #12819 (2 μg/ml, 8 hr) and XBP-1s (E8C2Z) Mouse mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 7 (upper), including DNAJB9 (lower), a known target gene of XBP-1s (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: XBP-1s (E8C2Z) Mouse mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells treated with tunicamycin #12819 (2 μg/ml, 8 hr) and either XBP-1s (E8C2Z) Mouse mAb #27901 or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human DNAJB9 Exon 1 Primers #79879, human HSPA5 promoter primers, human HNRNPA3 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.