For ResearchUseOnly. NotForUseInDiagnostic Procedures. ® 2015 Cell Signaling Technology, Inc. XP, SignalSilenceº and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc. Store at -20°C were labeledwithDyLight™554Phalloidin#13054 (red).Bluepseudocolor=DRAQ5 Dg—dog Species Cross-Reactivity Key: Applications Key: Confocal immunofluorescentanalysisofHeLa cells,transfectedwithSignalSilence Control siRNA protein. ing toresiduessurroundingThr270ofhumanSec24B by immunizinganimalswithasyntheticpeptidecorrespond Source/Purification: Monoclonalantibodyisproduced recognizes endogenouslevelsoftotalSec24Bprotein. Specificity/Sensitivity: Sec24B(D7D6S)RabbitmAb defects (8,9). mutations inSec24Bhavebeenlinkedtodevelopmental cells: Sec24A,Sec24B,Sec24C,andSec24D(7).Inmice, tion withSec23.TherearefourSec24isoformsinhuman the ERandbringsthemtoCOPIIvesiclesthroughinterac cargo selection(2,6).Itbindsdirectlytoproteinsat subunit ofCOPIIcoatisthoughttoplayacriticalrolein Sec13/31 toformmatureCOPIIcoat(4,5).TheSec24 molecules (1-3).Theprebuddingcomplexfurtherrecruits forming aprebuddingcomplexthatdirectlybindstarget activated Gprotein,Sar1,totheSec23/24complex,thereby (1). COPIIformationisinitiatedthroughthebindingof synthesized proteinsfromtheERtoGolgiapparatus ER/Golgi interfaceandisinvolvedintransportofnewly Sec13/31 complex,andSar1.COPIIcoatislocatedatthe composed offivecytosolicproteins:Sec23/24complex, Background: CoatProteinComplexII(COPII)is Sec24B siRNAI#12229(center),orSignalSilence #12042 0.1% Tween IMPORTANT: Forwesternblots,incubatemembrane withdilutedantibodyin5%w/vBSA,1XTBS, n Sec24B (D7D6S)RabbitmAb 3 Endogenous Applications W, IP, IF-IC Pg—pig  (10 westernblots) 100 µl ® Sc—S. cerevisiae 20 at4°Cwithgentle shaking,overnight. W—Western H—human Species Cross-Reactivity* IP—Immunoprecipitation Ce—C. elegans H, Mk M—mouse

Sec24B siRNAI ® Sec24BsiRNAII#12333(right),using (D7D6S)RabbitmAb(green).Actinfilaments Hr—horse R—rat IHC—Immunohistochemistry - - Hm—hamster Sec24B (D7D6S)RabbitmAb. Western blotanalysisofextractsfromvariouscelllinesusing Molecular Wt. 140 kDa 100 140 200 kDa 10 20 30 40 50 60 80 All—all species expected Mk—monkey ® ControlsiRNA(Unconjugated)#6568(left), SignalSilence ® #4084(fluorescentDNAdye). ChIP—Chromatin Immunoprecipitation Hep G2

293 Mi—mink Rabbit IgG** Sec24B siRNAII

HeLa rev. 01/05/15 Isotype

COS-7 Species enclosed inparenthesesarepredicted toreactbasedon100% homology. C—chicken Sec24B Dm—D. melanogaster IF—Immunofluorescence UniProt ID#O95487 Entrez-Gene ID#10427 Tween isaregisteredtrademark ofICIAmericas,Inc. subsidiaries. DyLight isatrademarkof ThermoFisherScientificInc.andits DRAQ5 isaregisteredtrademarkofBiostatusLimited. Background References: of recommendedcompanionproducts. Please visitwww.cellsignal.com foracompletelisting for thisproductatwww.cellsignal.com. For productspecificprotocolspleaseseethewebpage Immunofluorescence (IF-IC) 1:100 Immunoprecipitation 1:100 Western blotting Recommended AntibodyDilutions: ** *Species cross-reactivityisdeterminedbywesternblot. sodium azide.Storeat–20°C.Donotaliquottheantibody. mM NaCl,100µg/mlBSA,50%glycerolandlessthan0.02% Storage: Suppliedin10mMsodiumHEPES(pH7.5),150 (9)  (8)  (7)  (6)  (5)  (4)  (3)  (2)  (1)  detect thisantibody. antibodiesmustbeusedto Anti-rabbit secondary Wansleeben, C.etal.(2010)Development137,1067-73. Merte, J.etal.(2010)NatCellBiol12,41-6;suppp1-8. Tang, B.L.etal.(1999)BiochemBiophysResCommun258, Miller, E.etal.(2002)EMBOJ21,6105-13. Bi, X.etal.(2007)DevCell13,635-45. Barlowe, C.etal.(1994)Cell77,895-907. Mossessova, E.etal.(2003)Cell114,483-95. Miller, E.A.etal.(2003)Cell114,497-509. Aridor, M.etal.(1998)JCellBiol141,61-70. 679-84. X—Xenopus ® Support

Orders Web F—Flow cytometry Z—zebrafish n n n www.cellsignal.com [email protected] 877-678-TECH (8324) [email protected] 877-616-CELL (2355) E-P—ELISA-Peptide B—bovine

1:1000

kDa I II kDa 1 2 3 200 200 140 Sec24B 140 Sec24B 100 100 80 80 60 IgG heavy chain 50 60 50 40 30 40 30 20 50 β-Actin 40 10 – + + Sec24B siRNA – + – Rabbit (DA1E) mAb IgG XP® Isotype Control – – + Sec24B (D7D6S) Rabbit mAb Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) Immunoprecipitation of Sec24B from HeLa cell extracts, using #6568 (-), SignalSilence® Sec24B siRNA I #12229 (+), or Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or SignalSilence® Sec24B siRNA II #12333 (+), using Sec24B Sec24B (D7D6S) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Sec24B (D7D6S) Antibody #7427 (upper) or β-Actin (D6A8) Rabbit mAb #8457 Rabbit mAb. (lower). The Sec24B Antibody confirms silencing of Sec24B expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

® 2015 Cell Signaling Technology, Inc. ® 2015 Cell Signaling Technology, Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com