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M uniiainof Quantification nM. 3.3 , I b k 5 k 2 ] { 6 5 8 z 00 u oe does model Our 1000. 6 5 n o md was Smad3 for and , 4 nM. 149 and 1 ifrnersl in result difference ½ S s { 3 MM k 1 GOF t ~ 7 z . k 0 8 TGF z bv 25 above 5 5 0 nM. 403 b 6 19 R m This . 5 for m s 633 b { 6 R 1 uieaeasy rpoenextraction. protein or assays luciferase For 24– and for Immunohistochemistry develop to allowed were immunohistochemistry, embryos for processed Transfected and V. dissected ms hours, 40 50 48 delivering of electroporator was electroporation (TSS-100) pulses and Pulse neural tube square Dual neural 12 Intracel stage the an of HH using side of performed either lumen placed the were into electrodes injected tubes, was and (Hamburger DNA Hamilton Plasmid and 1951). Hamburger Hamilton, to according staged were embryos Chick vector. pCIG ovo a In into cloned were were Smad4 FLAG- Controls N-terminal and Human tube. Smad3 hairpin vector. neural Smad2, pSHIN short the tagged empty generate into the to electroporated electroporating 2004) fragments by be al., These performed could et protocol. (Kojima that vector (shRNAs) published pSHIN RNAs previously the into a cloned following were targeted were 283) chicken of sequences Non-overlapping Plasmids nlsdterslsadwoetemnsrp.EGG n S.P. and experiments, E.G.G. experiments. co-IP the manuscript. and WB the performed the wrote analysed and and and performed conceived conceived, results the E.M. analysed and D.G.M. assistance. contributions technical Author invaluable her for Usieto Susana to grateful are We Acknowledgements the quantifying first EGFP by of calculated number was cells electroporated differentiated of ratio The analysis data and Quantification sonication 1% glycerol, by 10% lysed NaCl, were mM cells 137 CaCl 7.4, The mM pH hpe. Tris-HCl, 1 24 mM DNA NP-40, at (20 buffer of dissected lysis pCIG, was amount in vector pulses tube total empty neural (the were the the indicated embryos with and DNAs co-immunoprecipitation, normalized the was by with complexes electroporated HH14 S234 at the of co-electroporated study the For blotting western and Immunoprecipitation a and sequences, promoter were Embryos CAGA hours (Promega). 24 and normalization firefly-luciferase after for ARE harvested a construct the reporter with by luciferase indicated Renilla driven DNAs the construct with reporter electroporated were Embryos vivo In niFA nioiswr eeae nhue(eata osLaboratory). Pons (Sebastian in-house generated were antibodies anti-FLAG and Anti-HA A-21271). Probes, (Molecular HuC/D ab28379), (Abcam, Smad3 5339), sqaiaieyhge hntemdlprediction. type model wild the the than with compared higher transcription qualitatively in is change fold where data, experimental rtaeihbtr 1m MF 1 PMSF, mM (1 inhibitors protease npsielssbfe.FrfyadRnlalcfrs ciiiswr measured were activities (Promega). luciferase system assay Renilla reporter homogenizer and luciferase Dounce Firefly dual a a with buffer. using ice lysis on passive homogenized and in dissected were tubes neural nirbi g yih 8 ojgt;Tem) h ebae eescanned were (Li-Cor). membranes imager The infrared Thermo). Odyssey conjugate; the 680 using and DyLight western conjugate and IgG 800 After antibodies DyLight anti-rabbit primary SDS-PAGE. IgG indicated (anti-Rabbit 8% the 2 antibodies by with secondary with incubated infra-red-tagged resolved boiled were were and membranes samples the times blotting, the three and washed buffer were loading beads The (Sigma). conjugate h ro asrpeettesem ttsia infcnei em of terms in significance Statistical and the s.e.m. value each using mean the calculated for represent the sections represent bars bars tube error The neural the S3). different Fig. three material cells (supplementary in electroporated condition cells 50 non-electroporated in 50 quantified versus was intensity change staining The embryos. immunofluorescence electroporated different neural in three the least of at from sections quantified transversal were confocal tube different ten least At electroporated. were rjc olwn tnadpoeue.Imnfursec a efre on 4 performed at hours was 2–4 Immunofluorescence procedures. (40 sections transverse standard RNA following whole-mount project for processed and rinsed situ PBS, in diluted paraformaldehyde muorcpttdfr4husa 4 at hours was volume 4 remaining the the for material; of input tenth the immunoprecipitated one as and reserved centrifugation was by supernatant removed resulting was material insoluble The Sigma). yrdzto sn rbsaantcikSa2adSa3fo h hce EST chicken the from Smad3 and Smad2 chick against probes using hybridization nsitu in electroporation uieaerpre assay luciferase-reporter ˚ sn h olwn oolnlatbde:Sa2(elSignaling, (Cell Smad2 antibodies: monoclonal following the using C yrdzto,ebyswr ie vrih t4 at overnight fixed were embryos hybridization, + el n hnb uniyn h HuC/D the quantifying by then and cells Z 2 ts ucini ubr (Macintosh). Numbers in function -test MMgCl mM 1 , m )atrfxto n4 aaomleyedltdi B for PBS in diluted paraformaldehyde 4% in fixation after m) novo in nsitu in n nyteitribscin fGFP-positive of sections interlimb the only and 2 .5 oimaie upeetdwith supplemented azide) sodium 0.05% , hybridization ˚ Smad3 m ihamncoa niH agarose anti-HA monoclonal a with C /lartnnad1 and aprotinin g/ml b 5–6)and 550–569) (bp + el mn hs that those among cells nsitu in n 5 m Smad2 e condition) per 5 /lleupeptin, g/ml hybridization, P vle was -values ˚ Cin4% b 264– (bp 6 SDS in Journal of Cell Science lre .C,Btetn .D n i,X. Liu, and D. M. Betterton, C., D. Clarke, aaa,D n eneg .A. R. Weinberg, and L. D. H. Hanahan, Hamilton, and V. Hamburger, Alarco R., R. Gomis, Garcı M. J. Gauthier, and S. Huet, P., Dijke, ten D., Vivien, S., Itoh, S., Dennler, X. Liu, and C. D. Clarke, eii . utnr .U,Prs .T,Ga,L . es,M,Kroa .S., T. Karpova, M., Reiss, R., L. Giam, T., W. Parks, U., J. Wurthner, A., Felici, M. J. Gauthier, and S. Huet, S., Dennler, M. Whitman, and J. M. Rubock, X., Chen, L. H. Cellie Moses, and A. J. Pietenpol, A., K. Brown, J. D. Bernard, ag . hn .R n Massague and R. C. Chen, Y., Kang, S. C. Hill, upeetr aeilaalbeoln at online available material Supplementary [grant EC the from D.G.M.]. a to to Grant and 248346-NMSSBLS S.P.] Reintegration BFU2011-30303 number to International numbers BFU2011-24099 Curie E.M.; [grant Marie to Government BFU2010-18959 Spanish D.G.M.; supported was the work This by position. RYC-2010-07450 a holds D.G.M. Funding un .R,Koc,C . nesn .C,Ilm . iof .K n Robertson, and K. E. Bikoff, A., Islam, C., D. Anderson, H., C. Koonce, R., N. Dunn, A. P. Merwe, der van and D. Coombs, O., Dushek, F., J. Allard, References http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.130435/-/DC1 i,S,Rn . i . hn,Y n eg A. Meng, and Y. 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