RNA was extracted from midguts of adult females 24 hr after blood feeding on a healthy

Transcription

1 Supplemental Data Cell Host & Microbe, Volume 5 The STAT Pathway Mediates Late-Phase Immunity against Plasmodium in the Mosquito Anopheles gambiae Lalita Gupta, Alvaro Molina-Cruz, Sanjeev Kumar, Janneth Rodrigues, Rajnikant Dixit, Rodolfo E. Zamora, and Carolina Barillas-Mury Experimental Procedures Cloning and sequencing of AgSTAT-A cdna RNA was extracted from midguts of adult females 24 hr after blood feeding on a healthy mouse. Mosquito mid-guts were dissected on ice-cold Ashburner-PBS, placed in ice-cold RNAlater (Ambion), and stored at 70ºC until mrna extraction. Poly(A) mrna was isolated from groups of 15 midguts using Oligotex-dT beads (QIAGEN), following the manufacturer s instructions. First-strand cdna was synthesized using random hexamers and Superscript II reverse transcriptase (Invitrogen). The AgSTAT-A cdna was cloned by using the cdna as template to amplify two overlapping PCR products of 2241 bp (F: GCGCCCAGTCGTGCTTCATTAGAG, all primer sequences are shown from 5' to 3' and R: GGACAGCGACCGGGTGGAGAA) and 647 bp (F: CTGGGTAAACGAGGGCAACGAC and R: TGCACCCCCACCCAGAGACACC). Primers were designed based on the cdna sequence predicted by the bioinformatic annotation of the STAT 2 gene in the An. gambiae genome sequence. These two fragments were sequenced and correspond to the regions between 767 to 3008 bp and 2921 to 3568, respectively, in the final cdna sequence (GenBank accession No. FJ and Figure S1). The 5' UTR region was cloned using the SMART RACE cdna amplification kit (Clontech; Mountain View, CA). A 2039 bp product was extended, amplified and cloned using a primer from the coding region (R:

2 GATGAACGTGTTGGTAATGAGC) and a 5'-end universal primer provided by the kit (F: CTAATACGACTCACTATAGGGCAAGAGTGGTATCAACGCAGAG). The sequence of this fragment corresponds to the region from 1 to 2039 bp in the final cdna sequence (Figure S1). Comparison of STAT sequences and phylogenetic analysis The predicted amino acid sequence of several members of the STAT family from different species (Hs = Homo sapiens, Dm = Drosophila melanogaster, Ct = Culex tritaeniorhynchus, Ae = Aedes aegypti, and Ag = Anopheles gambiae) were aligned and dendrograms constructed using the ClustalW software (Thompson et al., 1994), software is available at: The sequence alignment appears in Figure S2. The AgSTAT-A cdna sequence has been submitted to the GenBank accession No. FJ Quantitation of gene expression An. gambiae embryos were collected from 50 blood-fed females 12 hr after oviposition. Fourth instar larvae, light pupae and 3-day-old adult males and females were all collected in duplicate groups of 20 individuals each, frozen in liquid nitrogen and stored at 80 C. Poly(A) mrna was isolated using Oligotex-dT beads (QIAGEN), following the manufacturer s instructions. First-strand cdna was synthesized using random hexamers and Superscript II reverse transcriptase (Invitrogen). Gene expression was assessed by SYBR green quantitative real-time PCR (qpcr) (DyNAmo HS; New England Biolabs) in a Chromo4 system (Bio-Rad). PCR involved an initial denaturation at 95ºC for 15 min,

3 44 cycles of 10 s at 94 C, 20 s at 56 C, and 30 s at 72 C. Fluorescence readings were taken at 72 C after each cycle. A final extension at 72 C for 5 min was completed before deriving a melting curve (70 C 95 C) to confirm the identity of the PCR product. qrt-pcr measurements were made in duplicate. Relative quantitation results were normalized with An. gambiae ribosomal protein S7 as internal standard and analyzed by the 2 ΔΔCt method (Livak and Schmittgen, 2001) The following primers were used (5' to 3'): S7, F-AGAACCAGCAGACCACCATC, R-GCTGCAAACTTCGGCTATTC; AgSTAT-A, F-TACAACGAAACGACCAAGCA, R-GGTCCATACCGAAAAGACGA; AgSTAT-B, F- ACCGCGGCAACAGGAAACTAAA, R- GATAATGGTTGTCCATGCCAGTTG; SOCS, F-GTTTTCCGTCTCCTTCCGCAAGTA, R- CTTCGGTAGCGTCAGCTCGTTGAT; NOS, F- GCTCGAACTATCTGGCCAAC, R- CCACTCTTGCCAGAACGAAC; TEP1, F- CAGATGGTTCGTTTGGTGTG, R- GCAATGCCGTCAACACATAC. References Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22,

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5 CLUSTAL W (1.83) multiple sequence alignment AalSTAT MSLWARVNQLPQPVLEQIRYI-YGNSFPIEVRHYLAEWIEDRLL-NVPVYQHEQDSAYEQ 58 AaeSTAT MSLWARVNQLPQPVLEQIRYI-YGNSFPIEVRHYLAEWIEERLL-NVPVYQHEQDSAYEQ 58 CtSTAT MSLWARVNQLPPPVLEQIRYI-YGNSFPIEVRHYLAEWIEERLL-NAPVYQHDQDAAYEQ 58 AgSTATB MSLWARVNQLPQPILEQIRFI-YGSNFPIEVRHYLADWIEERLL-NAPVYTNDQEAVYEQ 58 AgSTATA --METRLHQLPPCILEQFHFL-NDLKYPVLIRQHLGNWIKDSLH-NAPTYTNNMQSMYEL 56 DmSTAT MSLWKRISSHVDCEQRMAAYY-EE-KGMLELRLCLAPWIEDRIM-SEQITPN-TTDQLER 56 AmSTAT MSLWAKAQQLPQDALQQVRSV-YGEHFPIEVRHFLSSWIEEKM--WTDIE--PDNPQYEQ 55 TcSTAT MSLWAKAQQLPPESLQQIRSI-YGDHFPIEVRHYLAHYIEEKF--WSDIDPVPDNPQHEQ 57 PmSTAT MSLWNRAQQLPADDLRRVQGI-YGEQFPIEVRHYLAGWIEDKMQQWNEID--PDNPSHSQ 57 HsSTAT5A MAGWIQAQQLQGDALRQMQVL-YGQHFPIEVRHYLAQWIESQP--WDAID--LDNPQDRA 55 HsSTAT5B MAVWIQAQQLQGEALHQMQAL-YGQHFPIEVRHYLSQWIESQA--WDSVD--LDNPQENI 55 HsSTAT6 MSLWGLVSKMPPEKVQRLY-----VDFPQHLRHLLGDWLESQP--WEFLVG--SDAFCCN 51 HsSTAT1 MSQWYELQQLDSKFLEQVHQLYDDS-FPMEIRQYLAQWLEKQDWEHAA NDVS 51 HsSTAT3 MAQWNQLQQLDTRYLEQLHQLYSDS-FPMELRQFLAPWIESQDWAYAA SKES 51 HsSTAT4 MSQWNQVQQLEIKFLEQVDQFYDDN-FPMEIRHLLAQWIENQDWEAAS NNET 51 HsSTAT2 MAQWEMLQNLDSPFQDQLHQLYSHSLLPVDIRQYLAVWIEDQNWQEAALG------SDDS 54. :* *. :::. AalSTAT EAATFLNQLINELERTAIN--LPEDNVTGRIRLNESARNFRQLFSHN--PVQ AaeSTAT EAATFLNQLINELERTAIN--LPEDNVTGRIRLNESARNFRQLFSHN--PVQ CtSTAT EAANFLNQLINELERTAIN--LPEDNVTGRIRLNESARNFRQLFSHN--PSQ AgSTATB DAANFLNQLIMELERTAIN--LPESNFTIKIRLNESARNFRQLFSHN--PAQ AgSTATA DAAKFFTALVNEVDQVSAN--LPN---KRKCLLCRSAIMLRDQNFQN--LTQ DmSTAT VALKFNEDLQQKLLSTRTA--SDQALKFRVVELCALIQRISAVELYT--HLRSGLQKELQ 112 AmSTAT YIATLVRSLIQELETKAAS-LNTDDMFLTKLKLMEAAKNFRQRYTHN--PAALFRIIR TcSTAT YVAGLVNSLIQEVENKAAV-VNDAEYFLTKLKLAEAAKMFRQRYRYNSNPMQLFSYVR PmSTAT YAQSLVSQLIQEIENKALSYANNEDLFLVRMRLDEAATSFRTRYLNS-NPLGLVGIIR HsSTAT5A QATQLLEGLVQELQKKAEH-QVGEDGFLLKIKLGHYATQLQKTYDRC--PLELVRCIR HsSTAT5B KATQLLEGLVQELQKKAEH-QVGEDGFLLKIKLGHYATQLQNTYDRC--PMELVRCIR HsSTAT6 LASALLSDTVQHLQASVGEQGEGS TILQHISTLESIYQRD--PLKLVATFR HsSTAT1 FATIRFHDLLSQLDDQYSRFSLE-NNFLLQHNIRKSKRNLQDNFQED--PIQMSMIIYS- 107 HsSTAT3 HATLVFHNLLGEIDQQYSRFLQE-SNVLYQHNLRRIKQFLQSRYLEK--PMEIARIVAR- 107 HsSTAT4 MATILLQNLLIQLDEQLGRVSKE-KNLLLIHNLKRIRKVLQGKFHGN--PMHVAVVISN- 107 HsSTAT2 KATMLFFHFLDQLNYECGRCSQDPESLLLQHNLRKFCRDIQP-FSQD--PTQLAEMIFN- 110.: : : AalSTAT LYSHLINCLQR----ERQCIAYPEECVNVQDPEIAEVINGLQQLQM 148 AaeSTAT LYSHLINCLQR----ERQCIAYPEECVNVQDPEIAEVINGLQQLQM 148 CtSTAT LYSHLINCLQR----ERQCIAYPNECVNVQDPEIAEVINGLQELQM 148 AgSTATB LYQHLMNCLHR----ERQCVAYPDECVNVQDPEVTEVFNAVQQLQI 148 AgSTATA LYLTLLHQIQPNC--EKGCKTEYTIAQTSSDGQQTDVLYGLQQLHV 145 DmSTAT LVTEKSVAATAGQSMPLNPYNMNNTPM--VTGYMVDPSDLLAVSNSCNPPVVQGIGPIHN 170 AmSTAT HCLGTEMKLVAQVENVGGTLMNMAGGKVGLISDAVAEIAQHVESLRR 157 TcSTAT QCLATEMRLVQAAN--GEALTGLAN---LVISNSGTEVLHKIDILRR 156 PmSTAT QCLNTEHNLVQQNE---NMLGGGVSHATNMVIEPCAEIEQELRILHE 158 HsSTAT5A HILYNEQRLVREAN----NCSSPAGILVDAMSQKHLQINQTFEELRL 153 HsSTAT5B HILYNEQRLVREAN----NGSSPAGSLADAMSQKHLQINQTFEELRL 153 HsSTAT QILQGEK HsSTAT CLKEERKILENAQRFNQ--AQSGNIQSTVMLDKQKELDSKVRNVKD 151 HsSTAT CLWEESRLLQTAATAAQQGGQANHPTAAVVTEKQQMLEQHLQDVRK 153 HsSTAT CLREERRILAAANMPVQG-PLEKSLQSSSVSERQRNVEHKVAAIKN 152 HsSTAT LLLEEKRILIQAQRAQLE--QGEPVLETPVESQQHEIESRILDLRA 154

6 AalSTAT LVRANENDNRNLVKDYEHLLLEIHEITKNKA QMEAIDNAQLREHARAALAQQ 200 AaeSTAT LVRANENDNRNLVKDYEHLLLEIHEITKNKA QMEAIDNAQLREHARAALVQQ 200 CtSTAT LVRVSENDNRNLVKDYEHLQLEIHEITKNKA LLENLDNAALREHARTTLAQQ 200 AgSTATB MVRTNENDNRNLMKEYEHLLLEVHELQKNRA QLETIENAEMRAHAHNQLAQH 200 AgSTATA MERNNWKETHQLIQECEQD--HVQRLSNQRS HYKRIQCYSLKQRS DmSTAT VQNTGIASPALGMVTPKVELYEVQHQIMQSLNE----FGNCANALKLLAQNYSYMLNSTS 226 AmSTAT RTQETGEDLRKMEQEQEAFAISYHECTKLNAHLQHFATQ-PQNQQNLDMEKKIRRQKEQQ 216 TcSTAT RTQDTADDLRRMEQEQESFALQYHECTKINAHIQQLSSQQPTNAQTTATVQKLTRQKEML 216 PmSTAT RTRETANELRHLEQEQESFALQYHDCAKINAHLSHIQSQ-ERTQQNREMEQSLRRRKELG 217 HsSTAT5A VTQDTENELKKLQQTQEYFIIQYQESLRIQAQFAQLAQLSPQERLSR--ETALQQKQVSL 211 HsSTAT5B VTQDTENELKKLQQTQEYFIIQYQESLRIQAQFGPLAQLSPQERLSR--ETALQQKQVSL 211 HsSTAT6 --KAVMEQFRHLPMP----FHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVS 161 HsSTAT1 KVMCIEHEIKSLEDLQDEYDFKCKTLQNRE HETNGVAKSDQKQE 195 HsSTAT3 RVQDLEQKMKVVENLQDDFDFNYKTLKSQGD MQDLNGNNQSVTRQK 199 HsSTAT4 SVQMTEQDTKYLEDLQDEFDYRYKTIQTMD QSDKNSAMVNQE 194 HsSTAT2 MMEKLVKSISQLKDQQDVFCFRYK-IQAKG KTPSLDPHQTKE 195. : AalSTAT ERNVNETVTLITGKRLNLVDNFRKTIQLTSQVQEKVLHKYLTQWKINQGFAGNGASGMSA 260 AaeSTAT ERNVNETVTLITGKRLNLVDNFRKTIQLTSQVQEKVLHKYLTQWKINQGFAGNGASGMSA 260 CtSTAT ERQVNEMVNLITGKRLQLVENFRKTIQLTSQVQEKVLHKYLTTWKINQGFAGNGAAGMSA 260 AgSTATB QKMVNDRLQLCTGKRLALVDGFRKTILITDEVQNKVLNKYLSQWKINQGFAGNGASMMSA 260 AgSTATA LVDAFQKTIRKAEEVLNLVYNKYIFEWQKTQMFP--EVRSTNA 229 DmSTAT SPNAEAAYRSLIDEKAAIVLTMRRSFMYYESLHEMVIHELKN-WRHQQAQAGNGAPFNEG 285 AmSTAT EQLLNHKVAGLMQLRLTLADKLKDTITRLNSLQSRVLDDELIRWKRDQQLGGNGAPF-NN 275 TcSTAT EQVLNQKVAGLMQLRLAIVDKFKETIQLLNQLQSNILDDELIRWKREQQLAGNGANF-NS 275 PmSTAT EQQLAQKVSGLLQLRMALADKHKGTIDRLNSLQQRILDEELINWKRDQQMHGNGKPF-NP 276 HsSTAT5A EAWLQREAQTLQQYRVELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPP-EG 270 HsSTAT5B EAWLQREAQTLQQYRVELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPP-EG 270 HsSTAT6 LHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLK-RIQIWKRQQQLAGNGAPF-EE 219 HsSTAT1 QLLLKKMYLMLDNKRKEVVHKIIELLNVTELTQNALINDELVEWKRRQQSACIGGPP--N 253 HsSTAT3 MQQLEQMLTALDQMRRSIVSELAGLLSAMEYVQKTLTDEELADWKRRQQIACIGGPP--N 257 HsSTAT4 VLTLQEMLNSLDFKRKEALSKMTQIIHETDLLMNTMLIEELQDWKRRQQIACIGGPL--H 252 HsSTAT2 QKILQETLNELDKRRKEVLDASKALLGRLTTLIELLLP-KLEEWKAQQQKACIRAPI--D 252 : *: * AalSTAT SNLDTIQAWCENLAEIIWNTKDQIRLAMKNKSKLNIEEPNLPDFLPQSLVEVTNLLKSLI 320 AaeSTAT SNLDTIQAWCENLAEIIWNTKDQIRLAMKNKSKLNIEEPNLPDFLPQSLVEVTNLLKALI 320 CtSTAT SNLDTIQAWCESLAEIIWNTKDQIRLAMKSKQKLNIEEPNLPDFLPQSLVEVTNLLKALI 320 AgSTATB SNLDTIQAWCESLAEIIWSTKDQIRLAIKNKSKLHVEQEDVPDLLPQAMVDVTNLLKMLI 320 AgSTATA YSLDEIQTWYESLAAIMWNTKDQIHLTMKSQLREHVSQEINSDLW-KVMKDVKDFIKLLL 288 DmSTAT S-LDDIQRCFEMLESFIAHMLAAVKELMRVRLVTEEPE------LTHLLEQVQNAQKNLV 338 AmSTAT N-LDSIQEWCESLAELIWLNRQQIKEAERLKQKFALEPPGMQDILPTLNSQITQLLSSLV 334 TcSTAT N-LDTIQDWCESLAELIWLNRQQIKEVDRLRQKLSLDPPGVADLLPQVLGDVTQLLSSLV 334 PmSTAT NKLDQIQEWCEALAEIIWLNRHQIKECERHQTKIPITPPGGVDMLPTLNSHITRLLSSLV 336 HsSTAT5A S-LDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPGP-VEEMLAEVNATITDIISALV 328 HsSTAT5B S-LDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPGP-VEEMLAEVNATITDIISALV 328 HsSTAT6 S-LAPLQERCESLVDIYSQLQQEVGAAG GELEPKTRASLTGRLDEVLRTLV 269 HsSTAT1 ACLDQLQNWFTIVAESLQQVRQQLKKLEELEQKYTYEHDPITKNKQVLWDRTFSLFQQLI 313 HsSTAT3 ICLDRLENWITSLAESQLQTRQQIKKLEELQQKVSYKGDPIVQHRPMLEERIVELFRNLM 317 HsSTAT4 NGLDQLQNCFTLLAESLFQLRRQLEKLEEQSTKMTYEGDPIPMQRTHMLERVTFLIYNLF 312 HsSTAT2 HGLEQLETWFTAGAKLLFHLRQLLKELKGLSCLVSYQDDPLTKGVDLRNAQVTELLQRLL 312 * :: : *.

7 AalSTAT TTTFIIEKQP PQVMKTNTRFAATVRLLIGNTLN-IRMSNPLVRVSIISEAQA 371 AaeSTAT TTTFIIEKQP PQVMKTNTRFAATVRLLIGNTLN-IRMSNPLVRVSIISEAQA 371 CtSTAT TTTFIIEKQP PQVMKTNTRFAATVRLLIGNTLN-IRMSNPLVRVSIISEAQA 371 AgSTATB TNTFIIEKQP PQVMKTNTRFAATVRLLVGNTLN-IKMVNPQVKVSIISEAQA 371 AgSTATA HKAFIVENQP PQVMKMNTRFCASVRLLIDNALI-MKIGNPKVTVSIISETQA 339 DmSTAT CSAFIVDKQP PQVMKTNTRFAASVRWLIGSQLG-IHNNPPTVECIIMSEIQS 389 AmSTAT TSTFIIEKQP PQVMKTNTRFTSTVRLLVGGKLN-VHMTPPQVKVSIISEAQA 385 TcSTAT TSTFIIEKQP PQVMKTNTRFTATVRLLVGGKLN-VHMTPPQVKVTIISESQA 385 PmSTAT TSTFIIEKQP PQVMKTNTRFTATVRLLVGGKLN-VNMTPPQVRVSIISEAQA 387 HsSTAT5A TSTFIIEKQP PQVLKTQTKFAATVRLLVGGKLN-VHMNPPQVKATIISEQQA 379 HsSTAT5B TSTFIIEKQP PQVLKTQTKFAATVRLLVGGKLN-VHMNPPQVKATIISEQQA 379 HsSTAT6 TSCFLVEKQP PQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQA 321 HsSTAT1 QSSFVVERQPCMPTHPQRPLVLKTGVQFTVKLRLLVKLQEL---NYNLKVKVLFDKDVNE 370 HsSTAT3 KSAFVVERQPCMPMHPDRPLVIKTGVQFTTKVRLLVKFPEL---NYQLKIKVCIDKDSGD 374 HsSTAT4 KNSFVVERQPCMPTHPQRPLVLKTLIQFTVKLRLLIKLPEL---NYQVKVKASIDKN HsSTAT2 HRAFVVETQPCMPQTPHRPLILKTGSKFTVRTRLLVRLQEG---NESLTVEVSIDRN *::: ** * ::* :* * *: : : : AalSTAT QATQQSNKASE------QSCGEIMNNTGNLEYNETTKQLSVSFRNMQLKKIKRAEKKGTE 425 AaeSTAT QATQQSNKASE------QSCGEIMNNTGNLEYNETTKQLSVSFRNMQLKKIKRAEKKGTE 425 CtSTAT QATQQSNKASE------QSCGEIMNNTGNLEYNETTKQLSVSFRNMQLKKIKRAEKKGTE 425 AgSTATB QQTQQTNKASE------QSCGEIMNNIGNLEYNETTKQLSVSFRNMQLKKIKRAEKKGTE 425 AgSTATA QQIQSTNAAAD------FSAGEIENNIGNLQYQLSNKFLAN-FSNMRLKKINRGNRKLNK 392 DmSTAT QRFVTRNTQMDNSSLSGQSSGEIQNASSTMEYQQNNHVFSASFRNMQLKKIKRAEKKGTE 449 AmSTAT NALLKSDKMAKN----GEASGEILNNTGTMEYHQATRQLSVSFRNMQLKKIKRAEKKGTE 441 TcSTAT NVLLKNDKLAKS----GECSGEILNNTGTMEYQQATRQLSVSFRNMQLKKIKRAEKKGTE 441 PmSTAT NALLKNDQMNK-----GEQSGEILNNTGTMEYNQTSRQLSVSFRNMQLRKIKRAEKKGTE 442 HsSTAT5A KSLLKNENTRN------ECSGEILNNCCVMEYHQATGTLSAHFRNMSLKRIKRADRRGAE 433 HsSTAT5B KSLLKNENTRN------DYSGEILNNCCVMEYHQATGTLSAHFRNMSLKRIKRSDRRGAE 433 HsSTAT6 RELSVPQGPGAG----AESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTE 377 HsSTAT1 RNTVKGFRKFNILG----THTKVMNMEESTNGSLAAEFRHLQLKEQK--NAGTRTNEGPL 424 HsSTAT3 VAALRGSRKFNILG----TNTKVMNMEESNNGSLSAEFKHLTLREQRCGNGGRANCDASL 430 HsSTAT4 VSTLSN-RRFVLCG----TNVKAMSIEESSNGSLSVEFRHLQPKEMKS-SAGGKGNEGCH 420 HsSTAT2 PPQLQGFRKFNILT----SNQKTLTPEKGQSQGLIWDFGYLTLVEQRSGGSGKGSNKGPL 422 :.. : AalSTAT SVMDEKFALLFQSSFAVGHGDLVFSVWTISLPVVVIVHGNQEPQSWATITWDNAFAD--I 483 AaeSTAT SVMDEKFALLFQSSFAVGHGDLVFSVWTISLPVVVIVHGNQEPQSWATITWDNAFAD--I 483 CtSTAT SVMDEKFALLFQSSFAVGHGDLVFSVWTISLPVVVIVHGNQEPQSWATITWDNAFAD--I 483 AgSTATB CVMDEKFALLFQSSFAVGHGDLVFSVWTISLPVVVIVHGNQEPQSWATITWDNAFAD--I 483 AgSTATA LVVDEKFALLFQSSFTLEQEELTVTVWTLSLPAVVIVHVNQEQLAWTTIIWDNLCAK--A 450 DmSTAT SVMDEKFALFFYTTTTVN--DFQIRVWTLSLPVVVIVHGNQEPQSWATITWDNAFAE--I 505 AmSTAT SVMDEKFSLLFQSQFSVGGGELVFAVWTLSLPVVVIVHGNQEPHAWATVTWDNAFAE--P 499 TcSTAT SVMDEKFSLLFQSQFSVGGGELMFQVWTLSLPVVVIVHGNQEPHAWATVTWDNAFSD--A 499 PmSTAT SVMDEKFSLLFQSQFSVGGGELVFQVWTLSLPVVVIVHGNQEPHAWATVSWDNAFAE--Q 500 HsSTAT5A SVTEEKFTVLFESQFSVGSNELVFQVKTLSLPVVVIVHGSQDHNATATVLWDNAFAE--P 491 HsSTAT5B SVTEEKFTILFESQFSVGGNELVFQVKTLSLPVVVIVHGSQDNNATATVLWDNAFAE--P 491 HsSTAT6 SVTEEKCAVLFSASFTLGPGKLPIQLQALSLPLVVIVHGNQDNNAKATILWDNAFSE--M 435 HsSTAT1 IVTEELHSLSFETQLCQPG--LVIDLETTSLPVVVISNVSQLPSGWASILWYNMLVAEPR 482 HsSTAT3 IVTEELHLITFETEVYHQG--LKIDLETHSLPVVVISNICQMPNAWASILWYNMLTNNPK 488 HsSTAT4 MVTEELHSITFETQICLYG--LTIDLETSSLPVVMISNVSQLPNAWASIIWYNVSTNDSQ 478 HsSTAT2 GVTEELHIISFTVKYTYQG--LKQELKTDTLPVVIISNMNQLSIAWASVLWFNLLSPNLQ 480 * :* : * : : : :** *:* : *. ::: * * AalSTAT NRVPFHVPDKVSWNLLAEALNTKFRASTG--RSMTPENMHFLCEKAFRAS-LQYPVSNDL 540

8 AaeSTAT NRVPFHVPDKVSWNLLAEALNTKFRASTG--RSMTPENMHFLCEKAFRAN-LQYPVSNDL 540 CtSTAT NRVPFHVPDKVSWNQLAEALNTKYRASTG--RSMTAENMHFLCEKAFRTN-LQFPVSDDL 540 AgSTATB NRIPFQVPDKVIWNQLAEALNMKFRASTG--RSLTAENMHFLCEKAFKTN-LPFPVPNDL 540 AgSTATA DRKLFEVPNLIPWNRLVEAISMTFSARVG--RGLTDENMQYMYRKAYRDK-LSFSVSNDQ 507 DmSTAT VRDPFMITDRVTWAQLSVALNIKFGSCTG--RSLTIDNLDFLYEKLQRE ERSE 556 AmSTAT GRVPFAVPDKVPWGQVAEALNVKFKSATG--RALTEDNLRFLAEKAFRGGNASGQDYSSL 557 TcSTAT GRVPFSVPDKVLWTNVAETLSLKFRAATG--RPLNEDNLRFLADKAFRGNYNEG---SNP 554 PmSTAT GRIPFTVPEKVPWPQIADMLDTKFKAATG--RGLTEDNLKFLAGKAFRNPQVQD--FTNM 556 HsSTAT5A GRVPFAVPDKVLWPQLCEALNMKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGL 551 HsSTAT5B GRVPFAVPDKVLWPQLCEALNMKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGL 551 HsSTAT6 DRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHR 495 HsSTAT1 NLSFFLTPPCARWAQLSEVLSWQFSSVTK--RGLNVDQLNMLGEKLLGPN----ASPDG- 535 HsSTAT3 NVNFFTKPPIGTWDQVAEVLSWQFSSTTK--RGLSIEQLTTLAEKLLGPG----VNYSGC 542 HsSTAT4 NLVFFNNPPPATLSQLLEVMSWQFSSYVG--RGLNSDQLHMLAEKLTVQS----SYSDG- 531 HsSTAT2 NQQFFSNPPKAPWSLLGPALSWQFSSYVG--RGLNSDQLSMLRNKLFGQN----CRTEDP 534 *. : :. : :. * : ::: : * AalSTAT AITWSQFCKEPLPDRTFTFWEWFYAAMKVTREHLRGPWNDGSIVGFIHKSKAEDYLLKCP 600 AaeSTAT TITWSQFCKEPLPDRTFTFWEWFYAAMKVTREHLRGPWNDGSIVGFIHKSKAEDYLLKCS 600 CtSTAT TITWSQFCKEPLPDRTFTFWEWFYAAMKVTREHLRGPWNDGSIVGFIHKTKAEDYLLKCQ 600 AgSTATB TIMWSQFCKEPIPDRSFTFWDWFYAAMKVTREHLRGPWMDGSIIGFIHKSKAEDYLLKCP 600 AgSTATA MISFAQFCKDTTPECNYTFWEWLYAALKIIRDHLQVLWVNNTIIGFIHKSTAEKYLAKCV 567 DmSTAT YITWNQFCKEPMPDRSFTFWEWFFAIMKLTKDHMLGMWKAGCIMGFINKTKAQTDLLRSV 616 AmSTAT LLSWAQFCKEPLPERNFTFWEWFYAVMKLTREHLKNPWVDGYILGFVRKRQAEEMLANCA 617 TcSTAT MLSWAQFCKEPLSERNFTFWEWFYAIMKLTREHLRGPWVDGAIIGFVKKKQAEEMLASCP 614 PmSTAT MLSWSQFCKEPLSERNFTFWEWFFAVMKVTREHLRQQWNDGSIMGFVGRRQAKEMLKNSK 616 HsSTAT5A SVSWSQFNRENLPGWNYTFWQWFDGVMEVLKKHHKPHWNDGAILGFVNKQQAHDLLINKP 611 HsSTAT5B SVSWSQFNRENLPGRNYTFWQWFDGVMEVLKKHLKPHWNDGAILGFVNKQQAHDLLINKP 611 HsSTAT6 SVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYVTSLLLNEP 555 HsSTAT1 LIPWTRFCKENINDKNFPFWLWIESILELIKKHLLPLWNDGCIMGFISKERERALLKDQQ 595 HsSTAT3 QITWAKFCKENMAGKGFSFWVWLDNIIDLVKKYILALWNEGYIMGFISKERERAILSTKP 602 HsSTAT4 HLTWAKFCKEHLPGKSFTFWTWLEAILDLIKKHILPLWIDGYVMGFVSKEKERLLLKDKM 591 HsSTAT2 LLSWADFTKRESPPGKLPFWTWLDKI : : * :.** *: AalSTAT R--GTFLLRFSDS-ELGGITIAWVNESNDG--QPQILHIQPFTAKDFATRSLSDRIR AaeSTAT R--GTFLLRFSDS-ELGGITIAWVNESNDG--QPQILHIQPFTAKDFATRSLSDRIR CtSTAT R--GTFLLRFSDS-ELGGITIAWVNESNDG--QPQILHIQPFTAKDFATRSLSDRIR AgSTATB R--GTFLLRFSDS-ELGGITIAWVNEGNDG--QPQILHIQPFTAKDFSTRSLSDRIR AgSTATA P--GTFLLRFTDS-VLGGISIAWVHESNDG--QRQVLHIQPFTAKDLVVRSLANRIC DmSTAT YGIGTFLLRFSDS-ELGGVTIAYVNENG------LVTMLAPWTARDFQVLNLADRIR AmSTAT S--GTFLMRFSDS-ELGGVTIAWVGDQT------EVFMLQPFTSKDFAIRSLADRVF TcSTAT C--GTFLLRFSDS-ELGGITIAWVSDTG------EVFSLQPFTSKDFAIRSLADRIA PmSTAT S--GTFLLRFSDS-ELGGVTIAWMYEDTTKACQRDVFMLQPFTSKAFAIRPLADVIA HsSTAT5A D--GTFLLRFSDS-EIGGITIAWKFDSP----ERNLWNLKPFTTRDFSIRSLADRLG HsSTAT5B D--GTFLLRFSDS-EIGGITIAWKFDSQ----ERMFWNLMPFTTRDFSIRSLADRLG HsSTAT6 D--GTFLLRFSDS-EIGGITIAHVIRGQDG--SPQIENIQPFSAKDLSIRSLGDRIR HsSTAT1 P--GTFLLRFSESSREGAITFTWVERSQNGG-EPDFHAVEPYTKKELSAVTFPDIIRNYK 652 HsSTAT3 P--GTFLLRFSESSKEGGVTFTWVEKDISG--KTQIQSVEPYTKQQLNNMSFAEIIMGYK 658 HsSTAT4 P--GTFLLRFSES-HLGGITFTWVDHSESG--EVRFHSVEPYNKGRLSALPFADILRDYK 646 HsSTAT AalSTAT DFEDLFYLYPNKPKNEAFDRYTTPPGQ-PRNRNYIPSEVRAVLMP---SNNNP 701 AaeSTAT DFEDLFYLYPNKPKNEAFDRYTTPPGQ-PRNRNYIPSEIYEFINH

9 CtSTAT DFEDLYYLYPNKPKNEAFDRYTSPPVP-SRNRNYIPSEVRAVLMGPSNNNNSS 704 AgSTATB DFDDLFYLYPNKPKHEAFDRYTTPAGP-PRNKNYIASEVRAVLMPG-PTNNQM 703 AgSTATA DLGELTYLYPTIPKQEAFGRYTAPAIQKPRSKHYISAEMRTVLIFAPSSNQSS 672 DmSTAT DLDVLCWLHPSDRNASPVKRDVAFGEFYSKRQEPEPLVL AmSTAT DLQHLLYLYPDISKDQAFSKYYTPFT--ENQSTSTNG TcSTAT DLNHLIYLYPDISKEIPFGKYYTPFQ--DNQPTSNNGYVKPVLVTH PmSTAT DLNYLLYLYPNVPKDQAFGKYYTPLG--EQQPTTNN HsSTAT5A DLSYLIYVFPDRPKDEVFSKYYTPV-----LAKAVDGYVKPQIKQVVPEF HsSTAT5B DLNYLIYVFPDRPKDEVYSKYYTPVPCESATAKAVDGYV HsSTAT DLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPE 660 HsSTAT1 VMAAENIPENPLKYLYPNIDKDHAFGKYYS-RPKEAPEPMELDGPKGTGYIKTELISVS- 710 HsSTAT3 IMDATNILVSPLVYLYPDIPKEEAFGKYC--RP-ESQEHPEAD--PGS HsSTAT4 VIMAENIPENPLKYLYPDIPKDKAFGKHYSSQPCEVSRPTERG---DKGYVPSVFIPIST 703 HsSTAT Figure S2. Sequence Alignment of the Predicted Protein Sequence of Several Members of the STAT Family of Transcription Factors from Different Species Hs = Homo sapiens, Dm = Drosophila melanogaster, Ct = Culex tritaeniorhynchus, Ae = Aedes aegypti, and Ag = Anopheles gambiae

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