For ResearchUseOnly. NotForUseInDiagnostic Procedures. ® 2014 Cell Signaling Technology, Inc. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. Store at -20°C additional applicationprotocols. See www.cellsignal.com forindividualcomponentapplications,speciescross-reactivity, dilutionsand Dg—dog Species Cross-Reactivity Key: Applications Key: and functionsasatranscriptional activator(11).PPARγ is member oftheligand-activatednuclearreceptor superfamily Peroxisome proliferator-activated receptorγ(PPAR γ) isa PGC1-α (10). non-histone proteinssuchasthetranscription co-activator protein foundinsimilarcomplexes(9).GCN5L2 acetylates to thep300/CBP-associatedfactorPCAF, anotherHAT coactivator complexes(8).GCN5L2is73%homologous catalytic subunitoftheSTAGA andTFTC transcription a histoneacetyltransferase(HAT) thatfunctionsas the Like 2(GCN5L2)isatranscriptionadaptorproteinand General ControlofAminoAcidSynthesisYeast Homolog histones andotherproteins(7). acetyltransferase (HAT) activity, allowingitto acetylate that associateswithPPARγ (6,7).CBPalsocontainshistone CBP (CREB-bindingprotein)isatranscriptionalco-activator blood flowviaeNOS/nNOS(5). growth throughEF2andTSC2/mTORpathways,aswell and glycogen,butalsomodulatesproteinsynthesiscell that AMPKnotonlyregulatesthemetabolismoffattyacids for AMPKactivation(2-4).Accumulatingevidenceindicates isrequired in theactivationloop,andthisphosphorylation AMPKαatThr172 tumor suppressorLKB1phosphorylates stress, suchasheatshock,hypoxia,andischemia(1).The elevated AMP/ATP ratioduetocellularandenvironmental (α1, 2;βγ2,3)(1).Thekinaseisactivatedbyan each ofwhichisencodedbytwoorthreedistinctgenes βandγsubunits, of acatalyticαsubunitandregulatory Background: antibody.western blotexperimentswitheachprimary tabolism. Thiskitincludesenoughantibodytoperformtwo evaluate PPARγ andrelatedproteinsinvolvedinlipidme Antibody SamplerKitprovidesaneconomicalmeansto Description: PPARγ RegulatedFattyAcidMetabolism Anti-rabbit IgG,HRP-linkedAntibody RXRα (D6H10)RabbitmAb SirT1 (C14H4)RabbitmAb PPARγ (C26H12)RabbitmAb GCN5L2 (C26A10)RabbitmAb CBP (D6C5)RabbitmAb AMPKα (D5A2)RabbitmAb Phospho-AMPKα (Thr172)(40H9)RabbitmAb Products Included #8660 n Antibody SamplerKit PPARγ RegulatedFattyAcidMetabolism 3 Pg—pig  (7 x20µl) 1 Kit AMPK isaheterotrimericcomplexcomposed Sc—S. cerevisiae W—Western H—human IP—Immunoprecipitation Ce—C. elegans M—mouse Hr—horse R—rat IHC—Immunohistochemistry - Product # 7074 3085 2496 2435 3305 7389 5831 2535 Hm—hamster respective totalproteins. (D6H10) RabbitmAballdetectendogenous levelsoftheir Rabbit mAb,SirT1(C14H4)and RXR mAb, GCN5L2(C26A10)RabbitPPARγ (C26H12) subunits. AMPKα(D5A2)RabbitmAb,CBP(D6C5) borγ catalytic subunit,butdoesnotdetecttheregulatory (40H9) RabbitmAbdetectsbothα1and2isoformsofthe at Thr172. Phospho-AMPKα(Thr172) phosphorylated (40H9) RabbitmAbdetectsendogenousAMPKαonlywhen Specificity/Sensitivity: Phospho-AMPKα(Thr172) scription duringlipidmetabolism(16). RXRs formheterodimerswithPPAR tohelpregulatetran- to thevitaminAderivative,9-cis-retinoicacid.Nuclear RXRβ, andγ)bindselectivelywithhighaffinity hormone receptorsencodedbythreedistinctgenes(RXR The humanretinoidXreceptors(RXRs)aretype-IInuclear fasting (14,15). liver andfatmobilizationinwhiteadipocytesresponseto 1α regulatesthegluconeogenic/glycolyticpathwaysin (PGC-1α) protein(15).DeacetylationofPPARγ andPGC- SirT1 includePPARγ (14),andthePPARγ coactivator-1α ing, glucosehomeostasis,aging,andlongevity. Targets of including apoptosis,cellularsenescence,endocrinesignal implicated intheregulationofmanycellularprocesses, SirT1, themammalianorthologofSir2,isanuclearprotein lases, alsoknownasclassIIIhistonedeacetylases(13). adenine dinucleotide(NAD)-dependentproteindeacety groupofgenesthatencodenicotinamide highly conserved The SilentInformationRegulator(SIR2)familyofgenesisa smooth musclecellsandmacrophage(12). preferentially expressedinadipocytesaswellvascular All—all species expected Quantity 100 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl Mk—monkey ChIP—Chromatin Immunoprecipitation 53, 57kDa Mol. Wt. 120 kDa 300 kDa 53 kDa 94 kDa 62 kDa 62 kDa Mi—mink Species enclosed inparenthesesarepredicted toreactbasedon100% homology. rev. 06/16 C—chicken Rabbit IgG Rabbit IgG Rabbit IgG Rabbit IgG Rabbit IgG Rabbit IgG Rabbit IgG Isotype Goat α Dm—D. melanogaster IF—Immunofluorescence - α, - U.S. PatentNo.5,675,063 Background References: PPARγ, SirT1andRXRαprotein. the respectivesequencesofhumanAMPKα,CBP, GCN5L2, AMPKα proteinorwithasyntheticpeptidecorrespondingto corresponding toresiduessurroundingThr172ofhuman by immunizinganimalswithasyntheticphosphopeptide Source/Purification: products. and acompletelistingofrecommendedcompanion Please visitwww.cellsignal.com forvalidationdata Western blotting Recommended AntibodyDilutions: sodium azide.Storeat–20°C.Donotaliquottheantibody. mM NaCl,100µg/mlBSA,50%glycerolandlessthan0.02% Storage: Suppliedin10mMsodiumHEPES(pH7.5),150 (16) (16) (15)  (14)  (13)  (12)  (11) (10)  (7)  (6)  (5)  (4)  (3)  (2)  (1)  (9)  (8)  Chan, H.M.andLaThangue,N.B.(2001) Goodman, R.H.andSmolik,S.(2000)GenesDev14, Hardie, D.G.(2004)JCellSci117,5479-87. Shaw, R.J.etal.(2004)ProcNatlAcadSciUSA101, Lizcano, J.M.etal.(2004)EMBOJ23,833-43. Hawley, S.A.etal.(1996)JBiolChem271,27879-87. Carling, D.(2004)Trends BiochemSci29,18-24. Yang, X.J.etal.(1996)Nature382,319-24. Candau, R.etal.(1996)MolCellBiol16,593-602. Gronemeyer, H.etal.(2004)NatRevDrugDiscov 3, Rodgers, J.T. etal.(2005)Nature434,113-8. Picard, F. etal.(2004)Nature429,771-6. Guarente, L.(1999)NatGenet23,281-5. Rosen, E.D.etal.(1999)MolCell4,611-7. Tontonoz, P. etal.(1995)CurrOpinGenetDev5,571-6. Lerin, C.etal.(2006)CellMetab3,429-38. 1553-77. 3329-35. 114, 2363-73. 950-64. X—Xenopus Support Orders Web F—Flow cytometry Z—zebrafish n n n Monoclonal antibodiesareproduced www.cellsignal.com [email protected] 877-678-TECH (8324) [email protected] 877-616-CELL (2355) E-P—ELISA-Peptide B—bovine 1:1000 J CellSci Western Immunoblotting Protocol #8660

For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for recommended primary antibody dilution buffer and recommended antibody dilution.

A. Solutions and Reagents C. Membrane Blocking and Antibody Incubations NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized mem- 1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml branes, adjust volumes accordingly. 20X PBS to 950 ml dH O, mix. 2 I. Membrane Blocking 2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room 900 ml dH2O, mix. temperature. 3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) 2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3. Wash three times for 5 min each with 15 ml of TBST. 3X SDS loading buffer. Dilute to 1X with dH2O. II. Primary Antibody Incubation 4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH O, mix. 1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as 2 recommended in the product datasheet) in 10 ml primary antibody dilution buffer with 5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X transfer buffer: add 100 gentle agitation overnight at 4°C. ml 10X transfer buffer to 200 ml methanol + 700 ml dH O, mix. 2 2. Wash three times for 5 min each with 15 ml of TBST. 6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X 3. Incubate membrane with the species appropriate HRP-conjugated secondary anti- TBST: add 100 ml 10X TBST to 900 ml dH O, mix. 2 body (#7074 or #7076 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 7. Nonfat Dry Milk: (#9999) 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with 8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat gentle agitation for 1 hr at room temperature. dry milk to 150 ml 1X TBST and mix well. 4. Wash three times for 5 min each with 15 ml of TBST. 9. Wash Buffer: (#9997) 1X TBST 5. Proceed with detection (Section D). 10. Bovine Serum Albumin (BSA): (#9998) 11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as indicated on primary antibody datasheet; for 20 ml, add 1.0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well. D. Detection of Proteins 12. Biotinylated Protein Ladder Detection Pack: (#7727) 1. Incubate membrane with 10 ml LumiGLO® (0.5 ml 20X LumiGLO® #7003, 0.5 ml 20X 13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720) peroxide, and 9.0 ml purified water) or 10 ml SignalFire™ #6883 (5 ml Reagent A, 5 ml 14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for Reagent B) with gentle agitation for 1 min at room temperature. nitrocellulose membranes. Pore size 0.2 µm is generally recommended. 2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and 15. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076) expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time. NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately 16. Detection Reagent: LumiGLO® chemiluminescent reagent and peroxide (#7003) or following incubation and declines over the following 2 hr. SignalFire™ ECL Reagent (#6883)

B. Protein Blotting A general protocol for sample preparation. 1. Treat cells by adding fresh media containing regulator for desired time. 2. Aspirate media from cultures; wash cells with 1X PBS; aspirate. 3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. 4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). 5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. 6. Microcentrifuge for 5 min. 7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm). NOTE: Loading of prestained molecular weight markers (#7720, 10 µl/lane) to verify electrotransfer and biotinylated protein lad- der (#7727, 10 µl/lane) to determine molecular weights are recommended. 8. Electrotransfer to nitrocellulose membrane (#12369).

LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories. Tween® is a registered trademark of ICI Americas, INC. SignalFire™ is a trademark of Cell Signaling Technology, INC.

Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com ® 2014 Cell Signaling Technology, Inc. ® 2014 Cell Signaling Technology,