Revision 1

#63734Store at -20C

1 Kit

(6 x 20 microliters)

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.
Product Includes Product # Quantity Mol. Wt Isotype/Source
TFEB (D2O7D) Rabbit mAb 37785 20 µl 65-70 kDa Rabbit IgG
Phospho-TFEB (Ser211) (E9S8N) Rabbit mAb 37681 20 µl 70 kDa Rabbit IgG
Phospho-TFEB (Ser122) Antibody 86843 20 µl 70-80 kDa Rabbit 
mTOR (7C10) Rabbit mAb 2983 20 µl 289 kDa Rabbit IgG
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb 5536 20 µl 289 kDa Rabbit IgG
Pan-Calcineurin A Antibody 2614 20 µl 59 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

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.

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.

Background References

    Trademarks and Patents

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

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    Revision 1
    #63734

    TFEB Signaling Antibody Sampler Kit

    TFEB Signaling Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using mTOR (7C10) Rabbit mAb.
    TFEB Signaling Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from Hela cells using mTOR (7C10) Rabbit mAb #2983. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
    TFEB Signaling Antibody Sampler Kit: Image 3 Expand Image
    Simple WesternTM analysis of lysates (0.1 mg/mL) from MCF-7 cells treated with insulin (100nM, 10 minutes) using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
    TFEB Signaling Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of lysates from MCF7, NIH/3T3, C6 and COS cells, using Pan-Calcineurin A antibody.
    TFEB Signaling Antibody Sampler Kit: Image 5 Expand Image
    Immunoprecipitation of mTOR protein from MCF-7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is mTOR (7C10) Rabbit mAb. Western blot analysis was performed using mTOR (7C10) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
    TFEB Signaling Antibody Sampler Kit: Image 6 Expand Image
    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 7 Expand Image
    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 8 Expand Image
    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 9 Expand Image
    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 10 Expand Image
    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 11 Expand Image
    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 12 Expand Image
    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 13 Expand Image
    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 14 Expand Image
    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 15 Expand Image
    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 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using mTOR (7C10) Rabbit mAb.
    TFEB Signaling Antibody Sampler Kit: Image 17 Expand Image
    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 18 Expand Image
    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 19 Expand Image
    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 20 Expand Image
    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 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using TFEB (D2O7D) Rabbit mAb.
    TFEB Signaling Antibody Sampler Kit: Image 22 Expand Image
    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 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using mTOR (7C10) Rabbit mAb.
    TFEB Signaling Antibody Sampler Kit: Image 24 Expand Image
    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 25 Expand Image
    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 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using TFEB (D2O7D) Rabbit mAb.
    TFEB Signaling Antibody Sampler Kit: Image 27 Expand Image
    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 28 Expand Image
    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.