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8660
PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit #8660

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PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 1

Immunohistochemical analysis of paraffin-embedded NCI-H228 cell pellets, control (left) or phenformin-treated (right), using Phospho-AMPKalpha (T172) (40H9) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 2

Western blot analysis of extracts from HeLa, K-562, C6, and Neuro-2a cells using AMPKα (D5A2) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 3

Western blot analysis of extracts from control HEK293T cells (Lane 1) or HEK293T cells with a targeted mutation in the gene encoding CBP (Lane 2) using CBP (D6C5) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The change in CBP molecular weight in the mutated HEK293T cells confirms the specificity of the antibody for CBP.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM, 1h) and CBP (D6C5) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across NR4A3, a known target gene of CBP (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of HeLa cells using GCN5L2 (C26A10) Rabbit mAb (green). Actin filaments have been labeled with DY554 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 6

Chromatin immunoprecipitations were performed with cross-linked chromatin from HDLM-2 cells and either PPARγ (C26H12) Rabbit mAb 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 FOXN3 Intron 3 Primers #95568, human STON2 intron 4 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.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 7

Western blot analysis of extracts from various cell types using SirT1 (C14H4) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 8

Chromatin immunoprecipitations were performed with cross-linked chromatin from LS-180 cells treated with Vitamin D (10nM) for 3 hours and either RXRα (D6H10) Rabbit mAb 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 c-Fos Upstream Primers #25661, human UCA1 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.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 9

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.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 10

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 11

Western blot analysis of extracts from various cell lines using CBP (D6C5) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 12

Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM, 1h) and CBP (D6C5) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 9 (upper), including NR4A3 (lower), a known target gene of CBP (see additional figure containing ChIP-qPCR data).

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 13

Western blot analysis of extracts from various cell lines using GCN5L2 (C26A10) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 14

Confocal immunofluorescent analysis of 3T3-L1 cells using PPARγ (C26H12A8) Rabbit mAb (red) showing nuclear localization in differentiated cells. Lipid droplets have been labeled with BODIPY 493/503 (green). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 15

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with Myc/DDK-tagged cDNA expression constructs encoding full-length human RXRα (hRXRα; +), RXRβ (hRXRβ; +), or RXRγ (hRXRγ; +), using RXRα (D6H10) Rabbit mAb (upper) and DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower).

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 16

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 17

Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells, treated with Forskolin #3828 (30 μM, 1h) and either CBP (D6C5) 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 human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, 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.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 18

Immunohistochemical analysis of 3T3-L1 cells, undifferentiated (left) or differentiated (right) , using PPARγ (C26H12) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 19

Western blot analysis of extracts from various cell lines using RXRα (D6H10) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 21

Confocal immunofluorescent analysis of HeLa cells using CBP (D6C5) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded mouse brown fat using PPARγ (C26H12) Rabbit mAb.

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 23

Western blot analysis of extracts from C2C12 cells, untreated or oligomycin-treated (0.5 µM), using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb (upper) or AMPKα Antibody #2532 (lower).

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit: Image 24

Western blot analysis of extracts from NIH/3T3 and 3T3-L1 cells (differentiated 6 days into adipocytes) using PPARγ (C26H12) Rabbit mAb.

To Purchase # 8660T
Product # Size Price
8660T
1 Kit  (7 x 20 µl) $ 514

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-AMPKα (Thr172) (40H9) Rabbit mAb 2535 20 µl
  • WB
  • IP
  • IHC
H M R Hm Mk Dm Sc 62 Rabbit IgG
AMPKα (D5A2) Rabbit mAb 5831 20 µl
  • WB
  • IP
H M R Mk B 62 Rabbit 
CBP (D6C5) Rabbit mAb 7389 20 µl
  • WB
  • IP
  • IF
  • ChIP
H M R Mk 300 Rabbit IgG
GCN5L2 (C26A10) Rabbit mAb 3305 20 µl
  • WB
  • IP
  • IF
H M R Mk 94 Rabbit IgG
PPARγ (C26H12) Rabbit mAb 2435 20 µl
  • WB
  • IHC
  • IF
  • ChIP
H M 53, 57 Rabbit IgG
SirT1 (C14H4) Rabbit mAb 2496 20 µl
  • WB
  • IP
H 120 Rabbit 
RXRα (D6H10) Rabbit mAb 3085 20 µl
  • WB
  • IP
  • ChIP
H M R 53 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

PPARγ Regulated Fatty Acid Metabolism Antibody Sampler Kit provides an economical means to evaluate PPARγ and related proteins involved in lipid metabolism. This kit contains enough primary antibody to perform two western blots per primary.

Specificity / Sensitivity

Phospho-AMPKα (Thr172) (40H9) Rabbit mAb detects endogenous AMPKα only when phosphorylated at Thr172. Phospho-AMPKα (Thr172) (40H9) Rabbit mAb detects both α1 and α2 isoforms of the catalytic subunit, but does not detect the regulatory β or γ subunits. AMPKα (D5A2) Rabbit mAb, CBP (D6C5) Rabbit mAb, GCN5L2 (C26A10) Rabbit mAb, PPARγ (C26H12) Rabbit mAb, SirT1 (C14H4) Rabbit mAb, and RXRα (D6H10) Rabbit mAb all detect endogenous levels of their respective total proteins.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr172 of human AMPKα protein or with a synthetic peptide corresponding to the respective sequences of human AMPKα, CBP, GCN5L2, PPARγ, SirT1 and RXRα protein.

Background

AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (1). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (1). The tumor suppressor LKB1 phosphorylates AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation (2-4). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (5).

CBP (CREB-binding protein) is a transcriptional co-activator that associates with PPARγ (6,7). CBP also contains histone acetyltransferase (HAT) activity, allowing it to acetylate histones and other proteins (7).

General Control of Amino Acid Synthesis Yeast Homolog Like 2 (GCN5L2) is a transcription adaptor protein and a histone acetyltransferase (HAT) that functions as the catalytic subunit of the STAGA and TFTC transcription coactivator complexes (8). GCN5L2 is 73% homologous to the p300/CBP-associated factor PCAF, another HAT protein found in similar complexes (9). GCN5L2 acetylates non-histone proteins such as the transcription co-activator PGC1-α (10).

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the ligand-activated nuclear receptor superfamily and functions as a transcriptional activator (11). PPARγ is preferentially expressed in adipocytes as well as in vascular smooth muscle cells and macrophage (12).

The Silent Information Regulator (SIR2) family of genes is a highly conserved group of genes that encode nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylases, also known as class III histone deacetylases (13). SirT1, the mammalian ortholog of Sir2, is a nuclear protein implicated in the regulation of many cellular processes, including apoptosis, cellular senescence, endocrine signaling, glucose homeostasis, aging, and longevity. Targets of SirT1 include PPARγ (14), and the PPARγ coactivator-1α (PGC-1α) protein (15). Deacetylation of PPARγ and PGC-1α regulates the gluconeogenic/glycolytic pathways in the liver and fat mobilization in white adipocytes in response to fasting (14,15).

The human retinoid X receptors (RXRs) are type-II nuclear hormone receptors encoded by three distinct genes (RXRα, RXRβ, and RXRγ) and bind selectively and with high affinity to the vitamin A derivative, 9-cis-retinoic acid. Nuclear RXRs form heterodimers with PPAR to help regulate transcription during lipid metabolism (16).

  1. Carling, D. (2004) Trends Biochem Sci 29, 18-24.
  2. Hawley, S.A. et al. (1996) J Biol Chem 271, 27879-87.
  3. Lizcano, J.M. et al. (2004) EMBO J 23, 833-43.
  4. Shaw, R.J. et al. (2004) Proc Natl Acad Sci USA 101, 3329-35.
  5. Hardie, D.G. (2004) J Cell Sci 117, 5479-87.
  6. Goodman, R.H. and Smolik, S. (2000) Genes Dev 14, 1553-77.
  7. Chan, H.M. and La Thangue, N.B. (2001) J. Cell Sci. 114, 2363-2373.
  8. Candau, R. et al. (1996) Mol Cell Biol 16, 593-602.
  9. Yang, X.J. et al. (1996) Nature 382, 319-24.
  10. Lerin, C. et al. (2006) Cell Metab 3, 429-38.
  11. Tontonoz, P. et al. (1995) Curr Opin Genet Dev 5, 571-6.
  12. Rosen, E.D. et al. (1999) Mol Cell 4, 611-7.
  13. Guarente, L. (1999) Nat Genet 23, 281-5.
  14. Picard, F. et al. (2004) Nature 429, 771-6.
  15. Rodgers, J.T. et al. (2005) Nature 434, 113-8.
  16. Gronemeyer, H. et al. (2004) Nat Rev Drug Discov 3, 950-64.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.