Neuro2a Mouse neuroblastoma cell line, about 30% of the cells grow like neuronal cells; 70% are round, loosely attached cells (DKMZ, Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH)
PC12 Htt25Q -EGFP PC12 Htt103Q
-EGFP Rat adrenal pheochromocytoma cell line; expressing a Htt exon 1 protein frag-ment containing either 25Q or 103Q fused to an EGFP-epitope tag after induc-tion with Muristerone A or Ponasterone A. Grow on collagene coated plates.
Differentiation by growing in starvation medium (1 % horse serum) supple-mented with 50 ng/ml NGF (Apostol et al., 2003).
HEK293 Human embryonal kidney cell line; adherent fibroblastoid cells growing as monolayer (DKMZ, Deutsche Sammulung für Mikroorganismen und Zellkul-turen GmbH).
4 .1 .3 Expression vectors
pcDNAI/Amp
-HD320_Q68 Expression vector for expression for the HD320_Q68 protein in mammalian cells under the control of the CMV promoter. The vector contains an ampicil-lin resistance.
pcDNA3.1-β-Gal Expression vector for the β-Galactosidase protein in mammalian cells under the control of the CMV promotor. Vector based on pcDNA3.1. The vector con-tains an ampicillin resistence.
pTL1-HA1-D48 Expression vector for HA-fusion proteins. Upstream of the multiple cloning site of pTL (1, 2, 3) the coding sequence for the HA-epitope has been intro-duced in all three reading frames (HA 1, 2, 3), and a Kozak-sequence has been inserted (Sittler et al., 1998) (Vector map: Appendix).
pc-myc-CMV-D12
Expression vector for the expression of fusion proteins containing an N-ter-minal Myc-epitope tag in mammalian cells under the control of the CMV promotor.Vector contains an ampicillin resistence (RZPD) (Vector map:
Appendix).
pPAReni-DM
pFireV5-DM Vectors are based on pCDNA3.1(+) (Clontech). For the pPAReni-DM the fol-lowing cassette was cloned between the BamHI and XbaI sites: Kozak sequence, a double protein A epitope, renilla luciferase and the ccdB cassette with flank-ing R1 and R2 att-sites. For the pFireV5-DM vector the followflank-ing cassette was cloned between the BamHI and XbaI sites: firefly luciferase , V5 epitope and the ccdB cassette with flanking R1 and R2 att-sites (Vector maps: Appendix).
pdECFP-Amp
pdEYFP-Amp Expression vectors for ECFP- and EYFP-fusion proteins based on pcDNA3.1 (+) (Clontec). Both vectors are Gateway-adapted destination vectors, containing the ccdB gene flanked by the attR recombination sites.
pdEYFP/pdECFP are designed for fusing a gene of interest to an N-terminal EYFP/ECFP-tag according to the Gateway Cloning Technology (Invitrogen) and for expression of this gene in mammalian cells under control of the hu-man CMV-IE promoter and enhancer (Vector maps: Appendix).
4 .1 .4 Microbiological media and buffers
1000x Ampicillin stock 100 mg/ml dissolved in 50% ethanol, stored at -20°C AttoPhos™ buffer 50 mM Tris-HCl pH 9.0,500mM NaCl, 1mM MgCl2 AttoPhos™ reagent 10 mM AttoPhos reagent dissolved in 100mM Tris pH9.0 1% BSA Bovine serum albumin was dissolved in 1x PBS to a final
concentration of 1 % (w/v)
Co-IP washing buffer 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.1 % NP-40, freshly added protease inhibitors (Complete™ final concentration: 1x; 1mM PMSF) 10x DNA sample buffer 0.42% bromophenol blue, 0.42% xylene cyanol, 25% ficoll type 400,
dissolved in distilled water, stored at -20°C
1000x Kanamycin stock 25mg/ml dissolved in distilled water, stored at -20°C Lysis buffer for mammalian
cells (native)
50mM Tris-HCl pH 8.8, 100 mM NaCl, 5mM MgCl2,
1%, NP-40 1mM EDTA, stored at 4°C, add protease inhibitors (Com-plete™ final concentration: 1x; 1mM PMSF) and 25U/ml benzonase straight before use
LB-(Luria Bertani) me-dium
10g/l Bacto Pepton, 5g/l yeast-extract, 10g/l NaCl, pH7.2
25x Protease inhibitors One tablet of Complete™ protease inhibitor (Roche) dissolved in 2ml distilled water, stored at -20°C
10x PBS 80g NaCl, 2g KCl, 14.4g Na2HPO4, 2.4g KH2PO4, to 1l with distilled water
4% PFA 4 g of paraformaldehyde dissolved in 100 ml PBS and heated at 50°C for 5 minutes to reach a clear solution, stored at -20°C PMSF 100mM PMSF dissolved in isopropanol, stored at -20.
12.5 % resolving gel 3.125ml of 40% Bisacrylamide, 2.5ml of 1.5 M Tris-HCl (pH8.8), 100μl of 10% SDS, 4.2ml ddH2O, 100μl of 10% APS,
and 10μl TEMED
5% stacking gel 0.75ml of 40% acrylamide, 1.5ml of 0.5M Tris-HCl (pH6.8), 60μl of 10% SDS, 3.6ml ddH2O, 60μl of 10% APS,
and 6μl TEMED
4x SDS loading buffer 200mM Tris-HCl pH6.8, 400mM DTT, 8% SDS, 4mg/ml bromophe-nol blue, 40% glycerol, store at -20°C
1 x SDS running buffer 1 x WB-buffer, 0.1% SDS
3% skim milk skim milk powder was dissolved in TBS-T to a final concentration of 3% (w/v)
Stripping buffer pH 2.2 200mM Glycin, 1% Tween20, 0.1% SDS
S.O.C. Medium 2% Tryptone, 0.5% Yeast Extract,10mM NaCl, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4, 20mM glucose
1x TBE 1x TBE 1mM EDTA, 45mM Tris-Borate pH 8.0
TBS 20mM Tris-HCl pH 7.5, 150mM NaCl
TBS-T 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.05% Triton X-100 10 x WB-buffer 30g/l Tris, 144g/l Glycin
Western blot buffer 1 x WB-buffer, 10 % Ethanol
4 .1 .5 Media and supplements for mammalian cell culture
Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/L D-Glucose, Sodium Pyruvate, without L-Glutamine
Gibco
Dulbecco’s modified Eagle medium (DMEM) with L-Glutamine, 1000 mg/L D-Glucose, Sodium Pyruvate
Gibco
0.5% Trypsin and 0.53mM Na-EDTA in Hanks’ B.S.S. Gibco Fetal calf serum (FBS) from E.G. approved countries Gibco Dulbecco’s phosphate buffered saline (D-PBS) without
calcium, magnesium
Gibco
L-Glutamine Gibco
Penicillin G (10000 units/ml) and
Streptomycin sulfate (10000μg/ml) in 0.85% saline
Gibco
MEM Non Essential Amino Acids (100X) Gibco
Geneticin (G418, 50mg/ml ) Gibco
Zeocin (100µg/ml) Invitrogen
Muristerone A (1mM in ethanol) Invitrogen/Sigma
4 .1 .6 Oligonucleotides
Table 4.1: Primer/probe quantitative real-time PCR
Name Sequence 5’ -> 3’
PC12LT_EGFP fw TGG TGA GCA AGG GCG AGG A
PC12LT_EGFP rev TGG TGC AGA TAG ACT TCA G
Sonde PC12LT_EGFP 6-FAM - CAG CCT GGT CGA GCT GGA - BHQ1
Oligonucleotides have HPLC purification grade and were synthesized by BioTeZ Berlin-Buch GmbH in a quantity of 10 nmol. Primers and probe have been resolved in nuclease-free water and diluted to a concentration of 10 pmol/µl.
siRNAs
siGENOME siRNAs were purchased from Dharmacon siRNA Technolgies (Thermo Scientific).
siGENOME smart pool siRNAs targeting genes of the p200 list were a kind gift of Dr. Steffan Wie-mann (DKFZ; Heidelberg).
siGENOME siRNA set of 4 and single siRNAs were purchased in a quantity of 5 nmol and have been resolved to a concentration of 20 µM in 1x siRNA buffer (Dharmacon; diluted from 5x siR-NA buffer in RsiR-NAse-free H2O).
Table 4.2: DDX24 targeting siRNA sequences
Name Target sequence
siDDX24_1 (D-097043-01) GGAAGCAAGUGACGAUCGA
siDDX24_2 (D-097043-02) ACUGACAGAUUACCGCUUA
siDDX24_3 (D-097043-03) GGUCGUAGCCUGGUAUUUG
siDDX24_4 (D-097043-04) ACGAAUCCUUCAUAAGAAA
4 .1 .7 Antibodies
Primary antibody Species Dilution Supplier
Anti-Actin mouse 1:2000 Abcam
Anti-CAG53b rabbit 1:5000 own production by E. Scherzinger
Anti-DDX24 rabbit 1:500 (WB)
1:100 (IF)
own production by M. Lalowski (antiserum)
Anti-GAPDH rabbit 1:2000 Sigma
Anti-HA (WB) mouse 1:2000 Babco
Anti-HA (IF) rabbit 1:100 Sigma
Anti-Myc mouse 1:100 Santa Cruz
Anti-PA rabbit 1:2000 Sigma
Anti-p53 rabbit 1:1000 Cell Signaling
Anti-V5 mouse 1:5000 Invitrogen
Secondary antibody Conjugate Species Dilution Supplier Anti-rabbit IgG Alkaline Phosphatase goat 1:10000 Sigma Anti-mouse IgG Alkaline Phosphatase goat 1:10000 Sigma
Anti-rabbit IgG Peroxidase goat 1:2000 Chemicon
Anti-mouse IgG Peroxidase goat 1:2000 Chemicon
Anti-rabbit IgG Cy3 donkey 1:200
Jackson Immuno Research
Anti-mouse IgG Cy5 goat 1:200 Dianova