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63734
TFEB Signaling Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

TFEB Signaling Antibody Sampler Kit #63734

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TFEB Signaling Antibody Sampler Kit: Image 1
Western blot analysis of lysates from MCF7, NIH/3T3, C6 and COS cells, using Pan-Calcineurin A antibody.
TFEB Signaling Antibody Sampler Kit: Image 2
Western blot analysis of extracts from 293, A431, COS, C6, and C2C12 cells, using mTOR (7C10) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 3
Western blot analysis of extracts from Raji, Daudi, and HDLM-2 cell lines, untreated (-) or treated with Torin 1 #14379 (250 nM, 5 hr), using Phospho-TFEB (Ser211) (E9S8N) Rabbit mAb (upper) or TFEB Antibody #4240 (lower).
TFEB Signaling Antibody Sampler Kit: Image 4
Western blot analysis of extracts from various cell lines using TFEB (D2O7D) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
TFEB Signaling Antibody Sampler Kit: Image 5
Western blot analysis of extracts from serum-starved NIH/3T3 cells, untreated or insulin-treated (150 nM, 5 minutes), alone or in combination with λ-phosphatase, using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (upper) or mTOR (7C10) Rabbit mAb #2983.
TFEB Signaling Antibody Sampler Kit: Image 6
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.
TFEB Signaling Antibody Sampler Kit: Image 7
Western blot analysis of extracts from GRANTA 519, REC-1, and Raji cells, untreated (-) or treated with Torin 1 #14379 (250 nM, 5 hr; +), using Phospho-TFEB (Ser122) Antibody (upper), TFEB (D2O7D) Rabbit mAb #37785 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
TFEB Signaling Antibody Sampler Kit: Image 8
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® mTOR siRNA II (+), using mTOR (7C10) Rabbit mAb #2983 and α-Tubulin (11H10) Rabbit mAb #2125. mTOR (7C10) Rabbit mAb confirms silencing of mTOR expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of mTOR siRNA.
TFEB Signaling Antibody Sampler Kit: Image 9
Western blot analysis of extracts from 293T cells, untransfected (-) or transfected with a construct expresssing full-length human TFEB (hTFEB; +) using TFEB (D2O7D) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 10
Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (#9904, 10 nM for 2 hours, left), insulin-treated (150 nM for 6 minutes, middle) or insulin- and λ-phosphatase-treated (right), using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
TFEB Signaling Antibody Sampler Kit: Image 11
Western blot analysis of extracts from GRANTA 519 cells, untreated (-) or treated with calf intestinal phosphatase and Lambda phosphatase (CIP/λ phosphatase; +), using Phospho-TFEB (Ser122) Antibody (upper), TFEB (D2O7D) Rabbit mAb #37785 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
TFEB Signaling Antibody Sampler Kit: Image 12
Confocal immunofluorescent analysis of Hela cells, using Pan-Calcineurin A Antibody exhibiting cytoplasmic localization (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
TFEB Signaling Antibody Sampler Kit: Image 13
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using mTOR (7C10) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 14
Immunoprecipitation of TFEB from Raji cell extracts. Lane 1 represents 10% input, lane 2 is immunoprecipitated with Rabbit (DA1E) mAb IgG XP® Isotype control #3900, and lane 3 is TFEB (D2O7D) Rabbit mAb. Western blot was performed using TFEB (D2O7D) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 15
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with constructs expressing Myc/DDK-tagged full-length human TFEB protein (hTFEB-Myc/DDK), Myc/DDK-tagged full-length human TFE3 protein (hTFE3-Myc/DDK), and Myc/DDK-tagged full-length human MITF protein (hMITF-Myc/DDK), using Phospho-TFEB (Ser122) Antibody (upper), Myc-Tag (71D10) Rabbit Ab #2278 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
TFEB Signaling Antibody Sampler Kit: Image 16
Flow cytometric analysis of NIH/3T3 cells, using Pan-Calcineurin A antibody (blue) compared to a nonspecific negative control antibody (red).
TFEB Signaling Antibody Sampler Kit: Image 17
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using mTOR (7C10) Rabbit mAb in the presence of control peptide (left) or mTOR Blocking Peptide #1072 (right).
TFEB Signaling Antibody Sampler Kit: Image 18
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using TFEB (D2O7D) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 19
Immunoprecipitation of Phospho-TFEB (Ser122) protein from GRANTA 519 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is Phospho-TFEB (Ser122) Antibody. Western blot analysis was performed using Phospho-TFEB (Ser122) Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
TFEB Signaling Antibody Sampler Kit: Image 20
Immunohistochemical analysis of paraffin-embedded mouse brain using mTOR (7C10) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 21
Immunohistochemical analysis of paraffin-embedded human lymphoma using TFEB (D2O7D) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
TFEB Signaling Antibody Sampler Kit: Image 22
Confocal immunofluorescent analysis of mouse embryonic fibroblast (MEF) cells using mTOR (7C10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
TFEB Signaling Antibody Sampler Kit: Image 23
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using TFEB (D2O7D) Rabbit mAb.
TFEB Signaling Antibody Sampler Kit: Image 24
Flow cytometric analysis of A549 cells using mTOR (7C10) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
TFEB Signaling Antibody Sampler Kit: Image 25
Chromatin immunoprecipitations were performed with cross-linked chromatin from Raji cells treated with Torin-1 (250 nM, 5 hr) and either TFEB (D2O7D) Rabbit mAb 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® Human GLA Promoter Primers #27131, human ATP6V1H 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.
To Purchase # 63734T
Cat. # Size Price
63734T
1 Kit  (6 x 20 microliters) N/A

Product Description

The TFEB Signaling Antibody Sampler Kit provides an economical means of analyzing the regulation of TFEB. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the TFEB Signaling Antibody Sampler Kit detects endogenous levels of its target protein. Pan-Calcineurin A Antibody detects endogenous levels of the alpha isoform of Calcineurin A. It may also recognize the beta and gamma isoforms of Calcineurin A. The antibody does not cross-react with protein phosphatase 1 or 2A.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Gly412 of human TFEB and Ser2481 of human mTOR, and synthetic phosphopeptides corresponding to residues surrounding Ser211 of human TFEB and Ser2448 of human mTOR. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide surrounding Ser122 and a synthetic peptide corresponding to the carboxy terminus of human Calcineurin A (alpha isoform). Antibodies are purified by protein A and peptide affinity chromatography.

Background

Transcription factor EB (TFEB) is a member of the Myc-related, bHLH leucine-zipper family of transcription factors that drives the expression of a network of genes known as the Coordinated Lysosomal Expression and Regulation (CLEAR) network (1,2). TFEB specifically recognizes and binds regulatory sequences within the CLEAR box (GTCACGTGAC) of lysosomal and autophagy genes, resulting in the upregulated expression of genes involved in lysosome biogenesis and function, and regulation of autophagy (1,2). TFEB is activated in response to nutrient deprivation, stimulating translocation to the nucleus where it forms homo- or heterooligomers with other members of the microphthalmia transcription factor (MiTF) subfamily and resulting in upregulation of autophagosomes and lysosomes (3-5). Recently, it has been shown that TFEB is a component of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which regulates the phosphorylation and nuclear translocation of TFEB in response to cellular starvation and stress (6-9). During normal growth conditions, TFEB is phosphorylated at Ser211 in an mTORC1-dependent manner. Phosphorylation promotes association of TFEB with 14-3-3 family proteins and retention in the cytosol. Inhibition of mTORC1 results in a loss of TFEB phosphorylation, dissociation of the TFEB/14-3-3 complex, and rapid transport of TFEB to the nucleus where it increases transcription of CLEAR and autophagy genes (10). TFEB has also been shown to be activated in a nutrient-dependent manner by p42 MAP kinase (Erk2). TFEB is phosphorylated at Ser142 by Erk2 in response to nutrient deprivation, resulting in nuclear localization and activation, and indicating that pathways other than mTOR contribute to nutrient sensing via TFEB (3).
Additional studies have also identified phosphorylation of TFEB at Ser122 that is dependent on mTORC1 (11). mTOR activity is associated with phosphorylation at Ser2448 via the PI3 kinase/Akt signaling pathway (12). Lysosomal calcium release activates the phosphatase calcineurin that dephosphorylates TFEB and promotes nuclear translocation and autophagy (13).
  1. Sardiello, M. et al. (2009) Science 325, 473-7.
  2. Sardiello, M. and Ballabio, A. (2009) Cell Cycle 8, 4021-2.
  3. Settembre, C. et al. (2011) Science 332, 1429-33.
  4. David, R. (2011) Nat Rev Mol Cell Biol 12, 404.
  5. Cuervo, A.M. (2011) Science 332, 1392-3.
  6. Peña-Llopis, S. et al. (2011) EMBO J 30, 3242-58.
  7. Settembre, C. and Ballabio, A. (2011) Autophagy 7, 1379-81.
  8. Peña-Llopis, S. and Brugarolas, J. (2011) Cell Cycle 10, 3987-8.
  9. Settembre, C. et al. (2012) EMBO J 31, 1095-108.
  10. Martina, J.A. et al. (2012) Autophagy 8, 903-14.
  11. Vega-Rubin-de-Celis, S. et al. (2017) Autophagy 13, 464-472.
  12. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  13. Medina, D.L. et al. (2015) Nat Cell Biol 17, 288-99.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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