Materials and Methods
1.3 DNA plasmids
pSG5
pSG5 is a eukaryotic cell expression vector with SV40 promoter.
pSG5-Rta, pSG5-Zta
pSG5-Zta is a B95.8 strain Zta-expressing plasmid. pSG5-Rta is a B95.8 strain Rta-expressing plasmid. Both of them use pSG5 as vector.
pEGFP-C1
pEGFP-C1 is a CMV promoter-driven green fluorescent protein (GFP)-expressing
15
plasmid with multiple cloning sites (MCS) located upstream of GFP.
pEGFP-Rta, pEGFP-Rta 1-441, pEGFP-Rta 401-605
Rta and truncated form of Rta plasmids are provided by Dr. Tuey-Ying Hsu.
Plasmids express B95.8 strain full-length Rta, amino acid 1 to amino acid 441 Rta (C terminal deletion), or amino acid 401 to amino acid 605 Rta (N terminal deletion).
Expressed proteins are fused with GFP at C terminus.
pCDNA3
pCDNA3 is an eukaryotic cell expression vector with CMV promoter.
pCDNA3-IQGAP2-HA
pCDNA3-IQGAP2-HA is a HA-tagged IQGAP2 expression plasmid using pcDNA3 as vector.
pGL2, pGL3
pGL2 and pGL3 (Promaga) are luciferase reporter vector lack eukaryotic promoter sequences. Their MCS locate upstream of luciferase for promoter insert.
pGL2-Zp, pGL2-Rp
pGL2 luciferase reporter plasmids that use either 221 /+21 Zta promoter or -711/+256 Rta promoter.
pGL2-pBLLF1
This pGL2 luciferase reporter plasmid that uses EBV BLLF1 (gp350/220) promoter,
16 provided by Dr. Mei-Ru Chen
pGL3-pIQGAP2 -853/+291, -642/+291, -523/+291, -167/+291, and -41/+291
These pGL3 luciferase reporter plasmids use indicated part of IQGAP2 promoter to drive transcription of luciferase.
pLKO.1-shLuciferase, pLKO.1-shIQGAP2-2, and pLKO.1-shIQGAP2-3
These are small hairpin RNA (shRNA)-producing plasmids containing sequences
targeting luciferase or IQGAP2. (Table 3) pCMVΔR8.91
pCMV-ΔR8.91 is the packaging plasmid containing gag, pol and rev genes for lentiviruses. (Purchased from Academia Sinica RNAi core)
pMD.G
pMD.G is the envelope plasmid that expresses G glycoprotein of vesicular stomatitis virus (VSV-G). (Purchased from Academia Sinica RNAi core)
1.4 Primers
Table 1, 2
1.5 Antibodies
Table 4
17 1.6 Chemicals and Reagents
Chemicals and Reagents Manufacturer
2-mercaptoethanol (2-ME) Sigma
Ammonium persulfate (APS) Sigma
Ampicillin Sigma
Bovine serum albumin (BSA) Roche
Bromophenol blue Sigma
Chloroform Merck
Calf intestinal alkaline phosphatase (CIP) New England Biolabs
Diethyl pyrocarbonate (DEPC) Sigma
Dimethyl sulfoxide (DMSO) Merck
Dipotassium phosphate (K2HPO4) Merck
Disodium phosphate (Na2HPO4) Merck
Deoxyribonucleotide triphosphate (dNTP) Protech
Dithiothreitol (DTT) Promega
Dulbecco's modified Eagle's medium (DMEM) Hyclone
Dual-Glo® Luciferase Substrate Promega
Ethanol Burnett
18
Ethidium bromide (EtBr) Sigma
Ethylenediaminetetraacetic acid (EDTA) J.T. Baker
Fast green Sigma
Fat-free skim milk Anchor
Fetal bovine serum (FBS) Hyclone
Ficoll-Paque GE Healthcare
G418 sulfate Merck
Glycerol J.T. Baker
Glycine Bio Basic
Hydrochloric acid (HCl) J.T. Baker
IGEPAL Sigma
Imidazole Usb
Kanamycin Sigma
LipofectamineTM 2000 Invitrogen
L-glutamine Sigma
Methanol J.T. Baker
M-MLV Reverse transcriptase Promega
Non-liposome transfection reagent II (NTR II) T-pro
Opti-MEM® Invitrogen
Penicillin:streptomycin solution Gemini
Peptone Usb
PhusionTM Hot Start High-Fidelity DNA Polymerase Finnzymes
Protein assay dye Biorad
ProZyme DNA polymerase Protech
19
Random primers Bionovas
RNase A Sigma
Rnasin RNase inhibitor Promega
Roswell Park Memorial Institute medium (RPMI) Hyclone
Potassium chloride (KCl) J.T. Baker
Potassium dihydrogen phosphate (KH2PO4) Merck
Propidium iodide Sigma
Cφmplete Protease inhibitor cocktail tablets Roche
Puromycin Sigma
Sodium acetate (NaOAc) Merck
Sodium azide Sigma
Sodium chloride (NaCl) J.T. Baker
Sodium deoxycholate Sigma
Sodium dodecyl sulfate (SDS) Merck
Sodium fluoride (NaF) Merck
Sodium hydroxide (NaOH) J.T. Baker
Sodium orthovanadate (Na3VO4) Sigma
T4 DNA ligase New England Biolabs
N,N,N',N'-Tetramethylethylenediamine (TEMED) Sigma
20
Tween 20 J.T. Baker
Western Lightning™ chemiluminescence reagent plus PerkinElmer
Xylene cyanol FF BDH
Yeast extract BD
1.7 Kits
Kit Manufacturer
Gel/PCR DNA fragments extraction kit Geneaid Presto™ High-speed plasmid mini kit Geneaid Neon® Transfection System 10 µL Kit Invitrogen
Plasmid maxi kit Qiagen
RNeasy Mini Kit Qiagen
1.8 Buffers
Phosphate-buffered saline (PBS) -EDTA (per liter)
EDTA 0.2 g, KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 1.15 g, add ddH2O to 1 L, autoclaved.
0.5% Trypan blue (per 100 ml)
Trypan blue 0.5 g, add PBS to 100 ml.
10X Phosphate-buffered saline (PBS) (per liter)
KCl 2.0 g, KH2PO4 2.4 g, NaCl 80.0 g, Na2HPO4 14.4 g, adjust to pH 7.4 with NaOH,
21 mix and add ddH2O to 1 L.
Lysogeny broth (LB) medium (per liter)
NaCl 10.0 g, Peptone 10.0 g, Yeast extract 5.0 g, adjust to pH 7.0 with NaOH, mix and add ddH2O to 1 L, autoclaved.
Terrific broth (TB) medium (per liter)
Glycerol 4 ml, KH2PO4 2.31 g, K2HPO4 12.54 g, Peptone 12.0 g, Yeast extract 24.0 g, mix and add ddH2O to 1 L, autoclaved.
50X Tris-acetate-EDTA (TAE) buffer (per liter)
Acetic acid 57.1 ml, pH 8.0 0.5M EDTA 100 ml, Tris 242.0 g, mix and add ddH2O to 1 L.
6X DNA loading dye (per 100 ml)
Glycerol 30 ml, Xylene cyanol FF 0.25 g, mix and add ddH2O to 100 ml.
4X Sample loading buffer (per 100 ml)
Bromophenol blue 0.4 g, Glycerol 40 ml, SDS 8.0 g, pH 6.8 1M Tris 20 ml, mix and add ddH2O to 100 ml, store at -20℃, supplemented with 2-ME 14.3 μl/ml freshly before use.
DEPC ddH2O (per liter)
DEPC 1.0 g, add ddH2O to 1 L, autoclaved.
Radioimmunoprecipitation assay (RIPA) buffer (per liter)
22
pH 8.0 0.5M EDTA 4 ml, NaCl 87.0 g, NaF 2.1 g, Na3VO4 0.018 g, 10% SDS 10 ml, Sodium deoxycholate 5.0 g, pH 7.4 1M Tris 50 ml, mix and add ddH2O to 1 L, supplemented with 50X Cφmplete protease inhibitor 40 μl/ml freshly before use.
10X Western blot running buffer (per liter)
Glycine 188.0 g, SDS 10.0 g, Tris 30.0 g, adjust to pH 8.3 with NaOH, mix and add ddH2O to 1 L.
10X Western blot transfer buffer (per liter)
Glycine 188.0 g, Tris 30.0 g, mix and add ddH2O to 1 l, supplemented with methanol 200 ml 1X transfer buffer freshly before use.
Western blot blocking buffer (per 100 ml)
Fat-free skim milk powder 4.0 g, add western blot washing buffer to 100 ml.
10X Western blot washing buffer (per liter)
NaCl 90.0 g, pH 7.4 1M Tris 100 ml, Tween 20 20 ml, mix and add ddH2O to 1 L.
Antibody dilution buffer (per 50 ml)
Skim milk 0.25 g, 20% Sodium azide 0.5 ml, add western blot washing buffer to 50 ml
Stripping buffer (per 100 ml)
10% SDS 20 ml, pH 6.8 1M Tris 6.68 ml, add ddH2O to 100 ml, supplemented with 2-ME 8.2 μl/ml freshly before use.
23 0.1% TritonX-100 (per 100 ml)
100 μl TritonX-100, add 1X PBS to 100ml.
Fractionation buffers
Buffer A
pH 7.9 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 1 % IGEPAL, supplemented with 1X protease inhibitor freshly before use.
Buffer B
pH 7.9 20 mM HEPES, 0.4 mM NaCl, 1 mM EDTA, 10% glycerol, supplemented with 1X protease inhibitor freshly before use.
Chromatin immunoprecipitation (ChIP) buffers
Cell lysis buffer
pH 8.0 5 mM HEPES, 85 mM KCl, 0.5% IGEPAL, supplemented with 1 mM DTT, 1X protease inhibitor, 0.1 mM PMSF freshly before use.
Nuclei lysis buffer
pH 8.0 50 mM Tris, 10 mM EDTA, 1% SDS, supplemented with 1 mM DTT, 1X protease inhibitor, 0.1 mM PMSF, 0.1 M NaF, 1 mM Na3VO4 freshly before use.
IP dilution buffer
pH 8.0 16.7 mM Tris, 167 mM NaCl, 1 mM EDTA, 1% Triton X-100,
supplemented with 1 mM DTT, 1X protease inhibitor, 0.1 mM PMSF, 0.1 M NaF, 1
24 mM Na3VO4 freshly before use.
Low salt wash buffer
pH 8.0 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL, 0.1% SDS, 1%
deoxycholic acid
High salt wash buffer
pH 8.0 50 mM Tris, 300 mM NaCl, 5 mM EDTA, 1% IGEPAL, 0.1% SDS, 1%
deoxycholic acid LiCl wash buffer
pH 8.0 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 300 mM LiCl, 1% IGEPAL, 0.1% SDS, 1% deoxycholic acid
Tris-EDTA buffer (TE buffer)
1 mM EDTA, pH 8.0 10 mM Tris Elution buffer
Fresh prepared with 50 mM NaHCO3, 1% SDS Proteinase K buffer
pH 7.5 50 mM Tris, 25 mM EDTA, 1.25% SDS 2. Methods