Supplemental Information
Immunoprecipitation and Glutathione S-Transferase (GST) pull-down
For immunoprecipitation, the NHF were first grown to 80% confluence, irradiated with 20 J/m2 UV and further
cultured for 4 h in fresh medium. The cells were then harvested and the whole cell extracts made with lysis
buffer [400 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl [pH8.0], protease inhibitor cocktail (1 mM PMSF,
10 mM Leupeptin, 10 mM Pepstatin)]. The cell lysates were pre-cleared with 30 μl of protein G plus/ protein A
agarose for 1 h at 4 ºC. The immunoprecipitation was carried out at 4 oC for 3 h by incubation of 1 μg anti-XPB,
anti-Sug-1, anti-p53 antibody or normal IgG with cell extracts containing 2 mg proteins, followed by addition of
30 μl of protein G plus/protein A agarose for another 2 h. Immunoprecipitates were collected and washed five
times with lysis buffer, resuspended in SDS sample buffer and boiled for 5 min at 95 ºC. The bound proteins
were detected for p53 protein by Western blotting.
To perform GST-pull down assay, the GST and its fusion proteins were expressed in E. coli BL21 strain.
The recombinant proteins were visualized on SDS/PAGE gel with Coomassie blue staining. Equal amount of
GST fusion proteins was immobilized on glutathione sepharose 4B beads and washed three times with lysis
buffer [50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1% (v/v) Triton X-100]. The loaded glutathione sepharose 4B
beads were incubated with whole cell extracts made from 20 J/m2 UV-treated NHF cells in lysis buffer. After
incubation at 4 oC for 1 h with rotation, the beads were washed three times with lysis buffer and boiled in SDS
sample buffer. The bound proteins were then separated and detected by SDS/PAGE followed by Western
blotting using indicated antibodies.
Cell Culture, transient transfection and luciferase report assay
The cells [NHF, LFS (MDAH041)], were maintained in DMEM supplemented with 10% fetal bovine serum.
About 18-20 h before transfection, the cells were plated at a density of 1 - 3×105 per 35 mm diameter dish (or 3
- 10×105 per 100 mm diameter dish for Western analysis). Transfections were carried out with FuGENE 6
transfection reagent according to the manufacturer’s recommendation. Approx. 12 h after transfection, the
DNA-FuGENE mix was removed and cells were treated with proteasome inhibitor MG132 (1 and 5 µM) or its
vehicle DMSO as control for an additional 12 h period. The cells were harvested for luciferase assay and
Western analysis. When needed, ts85 cells were plated at 5×106 per 100 mm dish, transfected for 24 h and the
cells divided into six 35-mm diameter dishes and transferred in triplicates to restrictive temperature 37 ºC or
permissive 32 ºC for another 24 h. In some experiments, transfection periods were extended to 24 or 48 h and
MG132 (10 µM) was added 10 h before harvesting. The results of relative luciferase units (RLU) of the enzyme
activity assay were standardized against the total cellular protein and normalized to the values obtained in
controls, or in the absence of MG132. All the transfection and reporter assay experiments were independently
performed at least three times each and error bars represent the SEM of triplicate data points.
Reverse transcription- polymerase chain reaction (RT-PCR)
NHF were either UV-irradiated at a dose of 10 J/m2 or non-UV irradiated and cultured for 12 h after irradiation.
The cells were then treated for another 8 h with 0, 5, or 25 µM MG132. After the MG132 treatment, the cells
were washed twice with cold PBS and total RNA were isolated with QIAGEN RNeasy Mini Kit (Qiagen Inc.,
Santa Clarita, CA), according to manufacture’s instruction. Reverse transcription was done using the
SuperScript III first strand-synthesis system from Invitrogen (Carlsbad, CA) in a 20 µl reaction volume using
the protocol provided by the manufacture. Briefly, 1 µg of total RNA, 50 µM Oligo (dT)20 and 10 mM of dNTP
mix were incubated in a 10 µl reaction volume at 65 ºC for 5 min followed by chilling on ice for 1 min. 25mM
MgCl2, 10× RT buffer, 0.1M DTT, RNaseOUT (40 U/µl) and SuperScript III RT(200 U/µl) were added for
cDNA synthesis and the reaction kept at 50 ºC for 50 min and terminated at 85 ºC for 5 min. For PCR detection,
2 µl of cDNA from each processed samples were subjected to PCR using specific primers for p21waf1, MDM2,
DDB2 and GAPDH using PuReTaq Ready-To-Go PCR beads (GE healthcare, UK). PCR was performed using
10 pmole of each primer and a PCR program with 94 ºC for 15”, 55 ºC (58 ºC for MDM2) for 30” and 72 ºC for
1 min and with 30 (35 for MDM2 and 28 for p21waf1) cycles. The RT-PCR products were run in 2% agarose gel
and the DNA levels were visualized and determined by AlphaImager 2000 Documentation & Analysis System.
The DNA sequences of used primers were as following: MDM2 (Human), 5’GATGAAAGCCTGGCTCTGTGTGT-3’ (forward) and 5’-TTCCAATAGTCAGCTAAGGA-3’ (reverse);
p21waf1, 5’-CCTCAAATCGTCCAGCGACCTT-3’ (forward) and 5’-CATTGTGGGAGGAGCTGTGAAA-3’
(reverse); DDB2, 5’-CCACCTTCATCAAAGGGATTGG-3’ (forward) and 5’CTCGGATCTCGCTCTTCTGGTC-3’ (reverse); GAPDH, 5’-GAAGGTGAAGGTCGGAGT-3’ (forward) and
5’-GAAGATGGTGATGGGATTTC-3’ (reverse).
ChIP assay
The ChIP procedure was essentially done as described by Suzanne T. Szak et al (Szak et al. 2001) with
appropriate modifications. The wild-type p53 containing MCF7 cells were grown to 80% confluence, UVirradiated and then treated with MG132 at 10 µM for the indicated time period. The MG132 containing growth
medium was aspired from the cells and replaced with a 1% formaldehyde-containing phosphate-buffered saline
(PBS) solution for 10 min at room temperature. The cross-linking was terminated by addition of glycine to a
final concentration of 0.125 M and incubation for 5 min. The cells were washed twice with cold PBS and
scraped in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, 1.0% NP40, 0.5%
deoxycholate, 0.1% SDS, 1 mM PMSF, and a protease inhibitor cocktail). The cell lysates were sonicated to
yield chromatin fragments with DNA length ranging between 500 to 2000 bp as assayed by agarose gel
electrophoresis. After removing the debris, chromatin extracts were collected and protein concentration was
determined by Bio-Rad assay.
For immunoprecipitation, chromatin extracts with 2 mg protein for each sample were pre-cleared with 20 µl
(bed volume) of protein A/G agarose beads at 4 ºC for 30 min. The agarose beads were pelleted by
centrifugation and the supernatants were recovered in a new tube. 2 µg of primary antibody (either Pab 1801 or
Pab 421 for p53, or Sug-1 or S1 for proteasome) and new protein A/G agarose beads were added to the pre-
cleared chromatin extracts and incubated overnight at 4 ºC with slow rotation. The immunoprecipitates were
washed successively with 1 ml of low salt buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 1%
triton X-100, 2 mM EDTA), high salt buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1% SDS, 1% triton
X-100, 2 mM EDTA), LiCl washing buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, , 1.0% NP40, 1.0%
deoxycholate, 1 mM EDTA) and twice with TE buffer. The DNA-protein complexes were eluted twice with
150 µl of IP elution buffer (1% SDS, 0.1 M NaHCO3). The eluents were pooled together and the cross-links
were reversed by adding NaCl (a final concentration 0.2 M) into the eluents and incubating them at 65 ºC for 5
h. The DNAs were recovered by proteinase K and RNase A digestion, followed by phenol/chloroform
extraction and ethanol precipitation.
The p21waf1–specific PCR reactions were performed using p21waf1 primers [(5’CCGCTCGAGCCCTGTCGCAAGGATCC-3’ (forward) and 5’-GGGAGGAAGGGGATGGTAG-3’ (reverse)]
and puReTaq Redy-To-Go PCR beads for 28 cycles, with each cycle consisting of 1-min 95 ºC denaturation, 1min 61 ºC annealing and 2-min 72 ºC extension. The PCR products were resolved using 8% polyacrylaminde
gels in 1× TBE buffer (100 mM tris-HCl pH 8.4, 90 mM boric acid and 10 mM EDTA). The gels were stained
with ethidium bromide and the relative DNA levels were determined by AlphaImager 2000 Documentation &
Analysis System.
Reference
Szak ST, Mays D and Pietenpol JA. (2001). Mol Cell Biol, 21, 3375-3386.