Supporting Methods
Reagents.
Unless otherwise stated, reagents were obtained from Sigma.Isolation of Intestinal Epithelial Cells.
The middle one-third of the small intestine was placed in ice-cold PBS and opened longitudinally, food debris was removed, and the tissue was minced. Samples were washed 3 times in 150 mM NaCl containing 1 mM DTT and were then resuspended in dissociation buffer containing 130 mM NaCl, 10 mM EDTA, 10 mM Hepes (pH 7.4), 10% FCS, and 1 mM DTT and incubated at 37°C for 45 min. The tubes were then shaken vigorously to liberate epithelial cells from the lamina propria. The epithelial cell suspension was carefully aspirated and washed in ice-cold PBS. Cell pellets were collected and snap-frozen in liquid nitrogen. DNA was extracted from cell pellets by digestion with proteinase K, followed by a phenol-chloroform-isoamyl alcohol extraction. Whole cell protein extracts were prepared by lysing pellets in 4°C buffer containing 150 mM NaCl, 20 mM Hepes (pH 7.6), 1.5 mM MgCl2, 0.2 mM EDTA, and 1% Triton X-100, supplemented with 1 mM DTT, 1:200 protease inhibitor mixture III (Calbiochem), 100 mM b -glycerophosphate, 1 mM NaF, and 1 mM sodium orthovanadate.Kinase Assay, Electrophoretic Mobility-Shift Assay (EMSA), and Western Blotting.
Ik B kinase (IKK) kinase assays, EMSAs, and IKK Western blots were performed as described (1). Supershift antibodies were from Santa Cruz Biotechnology. Antibodies against the following antigens were used for additional Western blots: p53, phospho-Ser-15 p53, Bcl-XL (Cell Signaling Technologies, Beverly, MA), Mdm-2, actin (Oncogene), and Bcl-2 (e-Bioscience, San Diego). Densitometry was used to quantify IKK kinase activity, NF-k B binding activity, and protein abundance.Real-Time RT-PCR.
RNA was extracted from tissues by using RNeasy kits (Qiagen, Valencia, CA), and cDNA was prepared by using Superscript II kits (Invitrogen). The abundance of mRNAs was compared between samples by real-time PCR as previously described (2), using primer pairs listed below.Further Analysis of the Ikkb Locus.
To assess recombination by real-time PCR, genomic DNA was amplified by using primers that anneal to intronic sequences flanking the loxP insertion regions (primers c and d, Fig. 2C). This reaction preferentially amplified Ikkb D DNA. To evaluate residual Ikkb by real-time PCR, a segment of genomic DNA lying between the upstream loxP site and exon 3 was amplified. Real-time PCR was performed on a 7700 Sequence Analyzer (Applied Biosystems) using SYBR-green reagents.Primer Sequences
1. Ikkb genotyping
Fig. 2A, primer a 5'-TGACCCGGGAATGAATAGCA-3', primer b 5'-GTCTTCAACCTCCCAAGCCTT-3'.
2. Vil-Cre genotyping
Forward 5'-AAGAGGAAGGTGTCCAATTTACTGA-3', reverse 5'-GCCTGGCGATCCCTGAA-3').
3. Ikkb D allele quantification.
Fig. 2A, primer c 5'-TAGTCCAACTGGCAGCGAATAC-3', primer d 5'-CGCCTAGGTAAGATGGCTGTCT-3'.
4. Residual Ikkb F quantification
Forward 5'-AAGATGGGCAAACTGTGATGTG-3', reverse 5'-CATACAGGCATCCTGCAGAACA-3'.
5. Preparation of Southern Probe
Forward 5'-GCCTTTTGATTTGCACGCACAGAAA-3', reverse 5'-CACAGTGCCCACATTATTTAGATAGG-3'.
6. Real-time RT-PCR analysis of mRNA expression. The following primers were used:
a. Ik Ba : forward 5'-CCAGAACAACCTGCAGCAGAC-3', reverse 5'-GCTCAGGATCACAGCCAGCTT-3'.
b. p53: forward 5'-AGATCCGCGGGCGTAAAC-3', reverse 5'-TCTGTAGCATGGGCATCCTTT-3'.
c. Cre: forward 5'-AAGAGGAAGGTGTCCAATTTACTGA-3', reverse 5'-GCCTGGCGATCCCTGAA-3'.
d. c-IAP1: forward 5'-GGAAATTGACCCTGCGTTATACAGA-3', reverse 5'-TCTCGGTCCATACACACTTTACACATT-3'.
e. c-IAP2: forward 5'-GGAAATTGACTCCACGTTATATGAAAACT-3', reverse 5'-GCTCTTCCAATGACAAGCCTGA-3'.
f. Gadd45b : forward 5'-TATTTGACAGCCCCCTCATC-3', reverse 5'-CCCAGAAGGTATCACGGGTA-3'.
g. Bcl-2: forward 5'-CCGGGAGAACAGGGTATGATAA-3', reverse 5'-CCCACTCGTAGCCCCTCTG-3'.
h. Bcl-XL: forward 5'-GGTCGCATCGTGGCCTTT-3', reverse 5'-TCCGACTCACCAATACCTGCAT-3'.