Flow cytometric analysis of A204 cells using BiP (C50B12) Rabbit mAb (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Confocal immunofluorescent analysis of HeLa cells using Calnexin (C5C9) Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from various cell lines, using Ero1-Lα Antibody.
Western blot analysis of extracts from various cell lines, using IRE1α (14C10) Rabbit mAb.
Confocal immunofluorescent analysis of NIH/3T3 cells using PDI (C81H6) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF wild-type cells treated with tunicamycin (2ug/ml) overnight, and CHOP (L63F7) Mouse mAb #2895 or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse ATF-3 Intron 1 Primers #13059, mouse CHOP promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using PERK (D11A8) Rabbit mAb.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Immunohistochemical analysis of paraffin-embedded human glioblastoma using BiP (C50B12) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Calnexin (C5C9) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using PDI (C81H6) Rabbit mAb.
Flow cytometric analysis of HeLa cells, treated with tunicamycin (2 µg/mL, 8 hr; green) or untreated (blue) using CHOP (L63F7) 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.
Western blot analysis of extracts from various cell lines using PERK (D11A8) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using BiP (C50B12) Rabbit mAb.
Western blot analysis of extracts from PANC1, HepG2 and A204 cells using Calnexin (C5C9) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lymphoma using PDI (C81H6) Rabbit mAb.
Confocal immunofluorescent analysis of A-204 cells, untreated (left) or tunicamycin-treated (right), using CHOP (L63F7) Mouse mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using BiP (C50B12) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse spleen using PDI (C81H6) Rabbit mAb.
Western blot analysis of extracts from C6 and A-204 cells, untreated or treated with thapsigargin (300 nM, 2 hours) or tunicamycin (24 μg/ml, 2 hours), using CHOP (L63F7) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using BiP (C50B12) Rabbit mAb in the presence of control peptide (left) or BiP Blocking Peptide #1084 (right).
Western blot analysis of extracts from various cell types using PDI (C81H6) Rabbit mAb.
Western blot analysis of extracts from various cell lines using BiP (C50B12) Rabbit mAb.
|BiP (C50B12) Rabbit mAb 3177||20 µl||
||H M||78||Rabbit IgG|
|Calnexin (C5C9) Rabbit mAb 2679||20 µl||
||H Mk||90||Rabbit IgG|
|Ero1-Lα Antibody 3264||20 µl||
|IRE1α (14C10) Rabbit mAb 3294||20 µl||
||H M R||130||Rabbit IgG|
|PDI (C81H6) Rabbit mAb 3501||20 µl||
||H M R Mk||57||Rabbit|
|CHOP (L63F7) Mouse mAb 2895||20 µl||
||H M R||27||Mouse IgG2a|
|PERK (D11A8) Rabbit mAb 5683||20 µl||
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
|Anti-mouse IgG, HRP-linked Antibody 7076||100 µl||
The ER Stress Sampler Kit contains reagents to investigate ER stress within the cell. The kit contains enough primary and secondary antibodies to perform two Western blot experiments per primary antibody.
Each antibody in the ER Stress Antibody Sampler Kit detects endogenous levels of its target protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu156 of human PERK protein, the sequence around Gly584 of human BiP, the sequence around His963 of human IRE1α, the sequence of human PDI and the sequence of human CHOP. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a sequence around Ala51 of human calnexin, the sequence around Leu218 of human Ero1-Lα, and the sequence of mouse MBTPS2. Antibodies are purified by protein A and peptide affinity chromatography.
Secretory and transmembrane proteins are synthesized on polysomes and translocate into the endoplasmic reticulum (ER) where they are often modified by the formation of disulfide bonds, amino-linked glycosylation and folding. The ER contains a pool of molecular chaperone proteins including calnexin, BiP and protein disulfide isomerase (PDI). Calnexin is an ER membrane, calcium-binding protein that retains newly synthesized glycoproteins inside the ER to ensure proper folding and quality control (1,2). Irregular protein folding within the ER increases BiP synthesis, which binds misfolded proteins to prevent them from forming aggregates and to assist them to refold properly (3).
PDI catalyzes the formation and isomerization of disulfide bonds required for a protein to reach its native state (4). Studies have found that the resident ER protein endoplasmic oxidoreductin-1 (Ero1) provides oxidizing potential to the ER in Saccharomyces cerevisiae (5). Ero1-Lα is an ER membrane-associated N-glycoprotein that promotes oxidative protein folding (6). Disruptions of ER homeostasis leads to the accumulation of unfolded proteins. The ER has developed an adaptive mechanism called the unfolded protein response (UPR) to counteract compromised protein folding (7). This is regulated by proteins such as the membrane-bound transcription factor protease site 2 (MBTPS2) and the serine/threonine kinase IRE1 (8-12). The PERK eIF2α kinase is an ER resident transmembrane protein that couples ER stress signals to translation inhibition. ER stress increases PERK activity, which phosphorylates eIF2α to reduce protein translation. PERK activation during ER stress correlates with autophosphorylation of its cytoplasmic kinase domain (13,14). Phosphorylation of PERK at Thr980 can serve as a marker for its activation status.
During ER stress, the level of CHOP expression is elevated and CHOP functions to mediate programmed cell death (15).
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