The RRPA knock-in allele was generated by homologous recombination in TC1 ES cells.

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1 Supplemental Materials Materials & Methods Generation of RRPA and RAPA Knock-in Mice The RRPA knock-in allele was generated by homologous recombination in TC1 ES cells. Targeted ES clones in which the neo-selection cassette was deleted were analyzed by both Southern and PCR analysis (Figure S1A), and positive clones were injected into blastocysts to obtain chimeras. We obtained many high-percentage chimeras, and upon breeding with wild-type B6 mice, these chimeras successfully transmitted the targeted allele to yield heterozygous mice. The heterozygous mice appeared normal and male and female heterozygotes were bred to obtain homozygous knock-in animals. The presence of the mutation in the homozygous knock-in embryos was confirmed by RT- PCR of the targeted region followed by DNA sequencing (Figure 1B). We also created heterozygous mice by injecting ES clones from which the neo-selection cassette had not been deleted. Heterozygous male mice were bred with female splicer mice that harbor a Cre transgene that is expressed in the eggs and deletes the floxed neo cassette at the one-cell stage. The offspring were screened for the targeted allele without the neo cassette and absence of the Cre transgene, since continuous expression of the Cre recombinase causes decreased cell growth, cytopathic effects, and chromosomal aberrations 12. The resulting neo - mouse lines were used in parallel with the lines generated from ES clones where the neo-marker had been deleted by transfection of cre-recombinase. The RAPA knock-in mice were created using similar experimental strategy. Gene Expression Profiling Total RNA was isolated from control or TSA-treated MEFs using RNeasy Mini Kit. RNA

2 was analyzed on Affymetrix GeneChip Mouse Genome 430A 2.0 Array at W.M. Keck Affymetrix GeneChip Core Facility (Yale University). Data analysis was performed and gene expression heat map was generated using GeneSpring GX 7.3 Expression Analysis software (Agilent Technologies) at W.M. Keck Biostatistics Resource (Yale University). Supplemental Figure Legends Supplemental Figure 1. (A) Genotyping of RRPA mutant allele. Southern blot analyses of genomic DNA from ES cells (upper left and upper middle panels) or from the progeny of heterozygous mice (upper right panel) are shown. PCR analyses of genomic DNA from mice (lower left and lower right panels) are displayed. (B) Eye development abnormalities in RRPA homozygous embryos at Ed13.5 (C) H&E staining of the eyes of wild-type and RRPA homozygous embryos. Supplemental Figure 2 (A) Sequence and location of five selected κb-binding sites upstream of the Pax6 gene that were analyzed using ChIP. (B) and (C). Quantitation of the semi-quantitative PCR signals in TSA-rescued Pax6 transcripts as presented in Figures 5D and 5E. Supplemental Figure 3 (A) Strategy to generate RAPA knock-in mice by homologous recombination. A BanI restriction enzyme site was coupled to the mutation in the targeted allele for PCR

3 analysis of the mutant allele. Restriction enzyme sites of and and the probes used for Southern blot analysis are indicated. (B) Genotyping of RAPA mutant allele. PCR analysis of genomic DNA from the progeny of heterozygous mice is shown. (C) Interaction between p65 and IκBα or IκBβ in RAPA MEFs detected by immunoprecipitation. (D) p65 localization in MEFs following TNFα (10 ng/ml; 30 min) stimulation detected by immunostaining. (E) RAPA knock-in fibroblasts are highly susceptible to TNFa induced apoptosis. MEFs from RAPA knock-in embryos were treated with the indicated amounts of TNFa for 24 hours and cell viability was measured by trypan blue exclusion. Wild-type, +/RAPA and RAPA/RAPA were labeled as, +/m and m/m, respectively.

4 Ghosh-Supplemental Figure 1 A probe A 12kb 10kb 4.9kb 2.6kb 4.9kb 2.6kb loxp-loxp 744bp 361bp,383bp PvuI B dpc13.5 C dpc15.5

5 Ghosh-Supplemental Figure 2 A Five selected κb-binding sites upstream of Pax6 gene B TSA(nM) Relative mrna Levels MEF MEF C TSA(nM) Relative mrna Levels p65-/- 3T3 + -p65 p65-/- 3T3 + p65-rrpa

6 Ghosh-Supplemental Figure 3 A R R P S D R cgg cgg cct tct gat cgc B p65 allele probe A kb 4.9 kb +/RAPA +/RAPA RAPA/RAPA RAPA/RAPA BanI R A P A D R cgg gca cct gct gat cgc targeted p65 allele R274A, S276A (RAPA) neo loxp loxp probe A 12 kb 2.6 kb TK BanI 744bp 352bp,392bp Cre-mediated excision recombined p65 allele R274A, S276A (RAPA) BanI R A P A D R cgg gca cct gct gat cgc loxp loxp 2.6 kb C WE IP p65 D MEF p65 RAPA/RAPA MEF p65 WE RAPA/RAPA IB control IκBβ IκBα TNFα 30 p65 E Viability(%) /m m/m TNF! (ng/ml)