Over-Expressionconstructs

METHODS

Over-expressionConstructs

pCAG-IP-HAwasconstructedbyannealingoligonucleotidescontaining3xHAwith SalIandNotIrestrictionsites atbothends intopCAG-IP(kindgift fromAustinSmith) thatwas digestedwithXhoIandNotI.AXhoIrestrictionsite is includeddownstreamof thetagsuchthatinsertscanbecloneintotheXhoIandNotIsites.Thesequencesofthe tagare:

5’TCGACATGTACCCATACGACGTCCCAGACTACGCTTACCCATACGACGTCCCAGACTACG CTTACCCATACGACGTCCCAGACTACGCTCTCGAGACCTTGGC-3’ and

5’GGCCGCCAAGGTCTCGAGAGCGTAGTCTGGGACGTCGTATGGGTAAGCGTAGTCTGGGA CGTCGTATGGGTAAGCGTAGTCTGGGACGTCGTATGGGTACATG-3’

Full-lengthEsetwithout stopcodonwas cloned frommouseembryonicstemcells cDNA

usingPfxDNApolymerase (Invitrogen)andthefollowingprimers:

5’-ATTGTCGACATGTCCTCCCTCCCTGGGTGCATG-3’and

5’-AGAATCGATAAGAAGTCTCCCTCTGCATTCAAT-3’

The resultingPCRproductwas clonedinto ZeroBlunt® TOPO®PCRCloningKit (Invitrogen)andsequencewereverified.TogeneratepCAG-IP-HA-ESET,alinkerwith astopcodon,Linker-ESETwith EcoRIandNotIstickyendswas ligatedwithpCAG-IP- HA digested with XhoIand NotIand TOPO-ESETdigested with SalIand EcoRI.For pCAG-IP-HA-control,a linkerwith stopcodon,Linker-controlwith SalIand NotIsticky ends was ligatedwithpCAG-IP-HAdigestedwithXhoIandNotI.

Sequences ofLinker-ESET are;

5’-AATTCTAACCGCGGGAGCTCGC-3’and5’-GGCCGCGAGCTCCCGCGGTTAG-3’. Sequences ofLinker-controlare;

5’-TCGACTAACCGCGGGAGCTCGC-3’and5’-GGCCGCGAGCTCCCGCGGTTAG-3’.

TogenerateHA-ESET-∆Tudor,pCAG-IP-HAdigestedwithXhoIandNotIwas ligated withpCAG-IP-HA-ESETdigestedwithNdeIandNotIandalinkerthatcontainsXhoI andNdeIstickyends;Linker-∆Tudor.

Sequences ofLinker-∆Tudorare;

5’-TCGAGATGTTCTGTTTGGATCCA-3’and5’-TATGGATCCAAACAGAACATC-3’.

TogenerateHA-ESET-∆SET,pCAG-IP-HAdigestedwithXhoIandNotIwas ligated withTOPO-ESETdigestedwith SalIandNdeIanda linkerthatcontainsastopcodon; Linker-∆SETwithNdeIandNotI stickyends.

Sequences ofLinker-∆Setare;

5’-TATGTTCTTGTTGACTAAGC-3’and 5’-GGCCGCTTAGTCAACAAGAACA-3’.

TogeneratepCAG-IG-Flag-Oct4,the followingprimers wereusedtoamplify fulllength Oct4frommouseembryonicstemcellscDNAandclonedintopCAG-IGatXhoIand NotIsites;

5’ATTGTCGACCACCATGGATTACAAGGATGACGACGATAAGATGGCTGGACACCTGGCTTC AGACTTCG-3’and5’-GTAGCGGCCGCTTAACCCCAAAGCTCCAGGTTCTCT-3’.

To generate pCAG-IG-Flag-Oct4-∆SIM, the following oligonucleotide primers were usedtosynthesis themutantstrandwithpCAG-IG-Flag-Oct4as thetemplate.

5’GGCTAGAGAAGGATGCGGCTCGAGTATGGTTCTG-3’and

5’CAGAACCATACTCGAGCCGCATCCTTCTCTAGCC-3’

TogeneratepCAG-IG-Flag-PML, the followingprimers wereusedtoamplify fulllength PmlfrommouseembryonicstemcellscDNA,andthe2.5kb productwasclonedinto pCAG-IGatXhoI andNotI sites;

5’ATTGTCGACCACCATGGATTACAAGGATGACGACGATAAGATGCCTCCCCCAGAGGAACC CTCCGAAG-3and’5’-GTAGCGGCCGCCTAGGCCAGGCATCCCTTACTTTCA-3’

To generate pCAG-IG-Flag-SUMO-1, the following primers were used to amplify

SUMO-1 fromhumanbraincDNAandclonedintopCAG-IGatXhoIandNotIsites;

5’ATTGTCGACCACCATGGATTACAAGGATGACGACGATAAGATGTCTGACCAGGAGGCAAA ACCTTCAA-3’and5’-GTAGCGGCCGCCTAACCCCCCGTTTGTTCCTGATAAACTTCA-3’