抗ATF6 (70B1413.1)抗体 | Anti-ATF6 (70B1413.1) Antibody

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	Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Analysis of ATF6. Lane 1, 293 cells transfected with full-length ATF6.* Lane 2, 293 cells transfected with partial length ATF6 (amino acids 1-373).* Lane 3, Untransfected 293 cells. Western blots were probed with 4 ug/ml of the ATF6 monoclonal antibody and visualized with PicoTect Western Blot Chemiluminescence Substrate (10087K). Film was exposed for 1 min. The top arrow corresponds to the ~90 kDa form of ATF6 described as full-length in the literature. *The human full-length and partial length ATF6 plasmids are described in Luo and Lee (2002).
	Biological Strategies Validation. Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Image of anti-ATF6. Whole cell protein from Hek293 and HepG2 treated with and without 10 mM DTT was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 5 ug/ml anti-ATF6 and detected with an anti-mouse HRP secondary antibody using a maximum sensitivity ECL reagent. Note the loss of the 90 kDa modified ATF6 protein (arrowhead) upon DTT treatment to activate the unfolded protein response.
	Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Analysis of ATF6 in mouse liver tissue using 3 ug/ml of ATF6 antibody and 0.25 ug/ml of GAPDH antibody. Lane A contains 20 ugs of whole mouse liver lysate, lane B contains 20 ugs of total ER fraction, and lane C contains 20 ugs of rough ER fraction. The ATF6 band may represent under glycosylated or cleaved/active ATF6.
	Immunocytochemistry/Immunofluorescence: ATF6 Antibody (70B1413.1) [NBP1-40256] - Untreated HeLa cells were fixed in -20C methanol for 10 min, air dried and rehydrated in PBS at room temperature for 5 minutes. Cells were incubated with anti-ATF6 (1:20) for one hour at room temperature. ATF6 reactivity (green) was detected with anti-mouse Dylight-488 secondary antibody. Nuclei were counterstained with DAPI (blue). Note the ER localization of ATF6.
	Immunohistochemistry-Paraffin: ATF6 Antibody (70B1413.1) [NBP1-40256] - ATF6 antibody (10 ug/ml) was used in IHC to stain formalin-fixed, paraffin-embedded human placenta, followed by biotinylated horse anti-mouse IgG secondary antibody, alkaline phosphatase-streptavidin and chromogen.
	Flow Cytometry: ATF6 Antibody (70B1413.1) [NBP1-40256] - Intracellular flow cytometric staining of 1 x 10^6 MCF-7 cells using ATF6 antibody (dark blue). Isotype control shown in orange. An antibody concentration of 1 ug/1x10^6 cells was used.
	Biological Strategies Validation. Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Image of anti-ATF6. Whole cell protein from HeLa cells treated with and without 10 mM DTT was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 5 ug/ml anti-ATF6 and detected with an anti-mouse HRP secondary antibody using a maximum sensitivity ECL reagent. Note the loss of the 90 kDa modified ATF6 protein (arrowhead) upon DTT treatment to activate the unfolded protein response.
	Flow (Intracellular): ATF6 Antibody (70B1413.1) [NBP1-40256] - An intracellular stain was performed on HeLa cells with ATF6 Antibody (70B1413.1) NBP1-40256F (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 10 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to FITC.
	Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Analysis of ATF6 in NIH3T3 cell lysate using NBP1-40256 at 3 ug/ml. A band corresponding to full-length ATF6 was detected. We have not characterized the ~36 kDa observed band; it may be an ATF6 breakdown/cleavage product.

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Background

ATF6 is a constitutively expressed, endoplasmic reticulum (ER) membrane-anchored transcription factor. ATF6 is a key transcriptional activator of the unfolded protein response (UPR), which allows mammalian cells to maintain cellular homeostasis when they are subjected to environmental and physiological stresses that target the ER (reviewed in Shen, 2005 & Prywes, 2005). The C-terminus of ATF6 is located in the ER lumen and its N-terminal DNA binding domain faces the cytosol. AFT6 plays a key role in the ER stress response by transmitting the ER stress signal across the ER membrane into the nucleus. The induction of new gene expression by ATF6 is an important aspect of the ER stress response. In response to certain stress conditions, ATF6 translocates from the ER to the Golgi. The 90 kDa full-length ATF6 is processed within the Golgi to its active 50 kDa form through sequential cleavage by site-1 and site-2 proteases (S1P and S2P). Proteolytic activation of ATF6 in the ER stress response is a mechanism to regulate membrane-bound factors, and is referred to as regulated intramembrane proteolysis. The N-terminal active ATF6 translocates to the nucleus where it binds to ER stress-response elements in ER stress-response genes (ERSRGs). ATF6 is a potent transcriptional activator of ERSRGs. The fully glycosylated form of ATF6, a 670 amino acid protein, exhibits an electrophoretic mobility of ~90 kDa in denaturing SDS-gels, in part because of the glycosylated modifications. ATF6 has 3 consensus sites for N-linked glycosylation and exists constitutively as a glycosylated protein. Differentially glycosylated ATF6 forms may result from mutations or experimental treatment.

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