The anti-nucleolin (catalog #sc-8031), anti – β-tubulin (catalog #sc-8035) monoclonal antibodies, as well as anti-MeCP2 (catalog #sc-20700) and anti – La (catalog #sc-33593) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-TatYB1 polyclonal antibody was raised as described previously (Capowski et al, 2001
). Anti-eFF1α monoclonal antibody (catalog #05-235) was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). Human anti-La/SS-B polyclonal antibody (catalog #L1380) was purchased from United States Biological (Swampscott, MA). SW1, SW3 and SW5.8 anti-La/SS-B monoclonal antibodies (Pruijn et al, 1995
) were a generous gift from Ger Pruijn.
Anti-rck/p54 polyclonal antibody was donated by Yukihiro Akao or purchased from MBL International (Woburn, MA; catalog #PD009). Anti-rck/p54 monoclonal antibody (DDX6 monoclonal antibody, clone 3D2) was purchased from Abnova Corporation (Taipei, Taiwan; catalog #H00001656-M01). Anti-PAI-RBP1 polyclonal antibody was a generous gift from Thomas Gelehrter. Anti-PAI-RBP1 monoclonal antibody was purchased from GeneTex (San Antonio, TX; catalog #GTX90457). Anti-APP polyclonal antibody was from Zymed Laboratories (South San Francisco, CA; catalog #51-2700). Anti-mouse β-actin antibody (catalog #A5441), mouse IgG (catalog #I5381) were purchased from Sigma Chemical Company (St. Louis, MO).
2.2. In vitro transcription
DNA oligonucleotides containing a T7 polymerase start site 5′ to APP specific sequences were generated by Integrated DNA Technologies. The APP specific sequences were as follows: 52sce FL: APP751 cDNA (accession #X06989) 2381 – 2432; Δ22: APP751 cDNA 2403 – 2432; sh52sce: APP751 cDNA 2403 – 2454. Oligonucleotides containing the reverse complement sequences were also generated. Forward and reverse sequences were annealed to generate double-stranded DNA templates for transcription. In vitro transcription was carried out with T7 RNA polymerase. Large quantities of RNA were generated with the MEGAscript T7 kit (Ambion). RNA was radiolabelled or biotinylated by including 32P-α-CTP (Amersham) or biotin-14-CTP (Invitrogen), respectively, in the transcription reaction. Transcription was carried out at 37°C for 2 hours. Transcripts were extracted with phenol-chloroform, precipitated with isopropanol, dissolved in diethyl pyrocarbonate-treated H2O, and quantified by absorbance at 260 nm.
2.3. Cell culture and lysates
SH-SY5Y cells were cultured on 12-well plates (Falcon) or on cover slips coated with Human Placental Collagen Type VI (Sigma) in 50/50 mix of Dulbeco's Modified Eagles Medium and Ham's F12 (50/50 DMEM/F12 mix; Gibco), supplemented with 1mM sodium pyruvate, 0.1mM L-glutamine, 50 μg/ml penicillin/streptomycin and 10% fetal bovine serum (FBS, Gibco) in a 5% CO2 atmosphere at 37°C. K562 cells were cultured in RPMI 1640 supplemented with 0.1mM L-glutamine, 50 μg/ml penicillin/streptomycin and 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37°C.
Cells were directly pelleted (K562 cells) or trypsinized and pelleted (SH-SY5Y cells), washed with DPBS and resuspended in IP buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 2.5 mM MgCl2, 0.1% NP-40, protease inhibitor cocktail, 1 mM DTT). Cells were lysed by swelling on ice for 10 min and the nuclei were pelleted at 600g for 20 min at 4 °C. The supernate fraction was collected as the cytosolic lysate. Lysates were used directly or stored at −80 °C.
2.4. Northwestern and immunoblotting
Proteins were denatured by incubation at 95 °C for 5 – 10 min in SDS sample buffer (4% SDS, 12% glycerol, 50 mM Tris (pH 6.8), 2% β-mercaptoethanol, 0.06% bromophenol blue) and electrophoresed on a Tris-Glycine-SDS polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane using the Trans-Blot Cell (Bio-Rad), according to the manufacturer's protocol.
For northwestern blotting, the membrane was incubated sequentially at room temperature in buffer NW (25mM HEPES, pH 8.0, 40mM KCl, 5mM MgCl2, 0.75mM DTT) containing 6M, 3M, 1.5M, 0.75M, 0.37M, 0.182M and 0M Guanidinium HCl. The membrane was blocked in buffer NW, 5% nonfat dry milk (NFDM) at room temperature for 90 min, and incubated at room temperature overnight with 107 cpm of radiolabelled RNA probe in buffer NW, 40U/ml RNasin Plus. The membrane was washed extensively in buffer NW, air-dried for 20 – 30 min, and imaged on a Storm® 860 Phosphorimager (Molecular Dynamics).
For immunoblotting, the membrane was blocked at room temperature for 1 hr in TBS-T (15mM TrisHCl, pH 7.6, 137mM NaCl, 0.1% Tween®-20), 5% NFDM, washed in TBS-T and incubated at room temperature for 240 min with a dilution of the primary antibody in TBS-T. Anti-nucleolin and anti-La/SS-B antibodies were diluted as per manufacturer's recommendations. Anti-rck/p54, anti-PAI-RBP1, anti-TatYB1 and anti-EF1α antibodies were biotinylated with the FluoReporter® Mini-Biotin-XX Protein Labelling Kit (Molecular Probes), according to the manufacturer's protocol. The biotinylated antibodies were diluted as follows for use in immunoblotting: 1:500 anti-TatYB1; 1:1000 anti-rck/p54; 1:500 anti-PAI-RBP1 and 1:1000 anti-EF1α. After incubation with the primary antibody, the membrane was washed in TBS-T, and incubated with a 1:1000 dilution of the appropriate HRP-conjugated secondary antibody, or 1:10000 dilution of peroxidase-conjugated streptavidin in TBS-T. Blots were imaged with ECL+ substrate on a Storm® 860 Phosphorimager (Molecular Dynamics).
2.5. RNA mobility shift assays
2.6. Protein purification and identification
K562 S100 lysate (900 μg) was pre-cleared by incubation at 37 °C for 30 minutes with 20 μg biotinylated 52sce FL RNA tethered to Dynabeads M-280 Strepavidin (Invitrogen) in Buffer L (10 mM Tris-HCl, pH 7.5, 0.1M KCl, 1mM DTT), 1X protease inhibitor cocktail, 0.1% bovine serum albumin, 2850 Units/ml RNasin Plus, for 30 min at 37 °C. Precleared lysate was incubated at 37 °C for 1 hr with 30 μg biotinylated sh52sce RNA tethered to Dynabeads M-280 Strepavidin, under the same buffer conditions. Dynabeads were washed with Buffer L, and proteins were eluted for 30 min at 37 °C with 300 μg free sh52sce RNA. A second elution with one bed volume of 2x SDS sample buffer (8% SDS, 24% glycerol, 100 mM Tris (pH 6.8), 4% β-mercaptoethanol, 0.12% bromophenol blue) was carried out at 95 °C for 5 min. Eluates were subjected to the Northwestern Blot procedure with the sh52sce radiolabelled RNA as probe. Active bands were excised out of an identically ran SDS-PAGE gel stained with Coomassie Brilliant Blue and subjected to MALDI-MS Peptide Mass Mapping at the Columbia University Protein Chemistry Core Facility (Columbia University, New York, NY). Peptide mass values were queried against the Mascot protein sequence database (Matrix Science, www.matrixscience.com
2.7. Immunoprecipitation, RNA isolation, cDNA synthesis and PCR analysis of APP mRNA
SH-SY5Y cell cytosol from 2X107
cells was pre-cleared with protein G Dynabeads in IP buffer containing phosphatase inhibitors ( 0.1mM Na3
, 10mM NaF, 20mM α-glycerophosphate) and 1000U/ml RNasin Plus. Pre-cleared lysate was incubated with 5 μg antibody and 50 μl Protein G Dynabeads (stock suspension volume) at 4°C for 5 hours. For La/SS-B immunoprecipitation, 50 μl Protein G Dynabeads (stock suspension volume) were pre-incubated for 1 hour at 4 °C with 50 μl SW1 and 50 μl SW5.8 hybridoma (Pruijn et al, 1995
) supernates; pre-cleared SH-SY5Y cytosol was mixed with the antibody-bound beads and incubated as above. Beads were washed 5 times with IP buffer and a portion of the IP reaction was transferred to TRI-Reagent (MRC, Inc.), total RNA isolated and precipitated in the presence of 50 μg glycogen. The final pellet was solubilized in diethyl pyrocarbonate treated H2
O and reverse-transcribed with Qiagen Omniscript and random nonamer primer (60 min at 37 °C, 5 min at 93 °C). The cDNA was PCR-amplified [15 min at 95 °C, (40 cycles: 30 sec at 95 °C, 1 min at 68 °C, 1 min at 72 °C), 10 min at 72 °C] with HotStarTaq DNA polymerase (Qiagen) and primers against the human APP gene (accession #X06989; forward: 899 – gacgaaccctacgaagaagcca – 920 and reverse: 1248 – gctttctggaaatgggcatgttc – 1226) which span the alternatively spliced site in the APP cDNA sequence. PCR reactions were analyzed on 1.5% agarose gels containing EtBr. Alternatively, cDNA was subjected to real time PCR with APP ORF FWD and APP ORF REV primers as described below. The amount of APP signal, n
= 3, was quantified and normalized to that seen in the pre-immune mouse IgG immunoprecipitate, ± S.E.M.
2.8. Plasmid preparation
For pTAT-HA-RCK plasmid preparation, the open reading frame of rck/p54 (NM_004397 bases 367-1785) was amplified with primers designed to add a unique NcoI
site to the 5′ end, and a unique XhoI
site to the 3′ end. The PCR product was digested with NcoI
and ligated to NcoI/XhoI
digested pTAT-HA vector (Vocero-Akbani et al, 2000
), which upon translation produces rck/p54 with N-terminal TAT and HA tags.
For pIRES-RCK-hrGFPII plasmid preparation, the open reading frame of rck/p54, except for the stop codon (NM_004397 bases 367-1782) was amplified. The forward primer was designed to add a unique BamHI site, followed by a consensus Kozak sequence (GCC ACC) immediately 5′ to the ATG start codon (NM_004397 bases 367- 369). The reverse primer was designed to generate a unique NotI site 3′ to the rck/54 open reading frame. The PCR product was digested and ligated to BamHI/NotI digested pIRES-hrGFP II plasmid (Stratagene), to produce pIRES-RCK-hrGFP II, which upon transcription produces a bicistronic mRNA with rck/p54 ORF followed by an internal ribosome entry site and an hrGFP ORF.
Rck/p54 insert sequence and in-frame insertion were verified by DNA sequencing at the UW Biotech Center DNA Sequencing Facility (University of Wisconsin – Madison, Madison, WI). The sequencing primers used were as follows: Rck/p54 (accession #NM_004397): 367 – atgggtctgtccagtcaa - 384; 867 - acaggtcagtcaaatttg – 884; 1367 – agcgagttgaattgctag – 1384; 617 – taatcttcaaactcatttc – 600; 1117 – tagcgtgagaataatatc – 1000; 1617 – gctaagccaagatgacca – 1600.
2.9. His-tagged protein purification and transduction
For purification of His-tagged TAT-RCK and TAT-GFP, BL21 strain E. coli
transformed with the appropriate plasmid were grown and induced with 1mM IPTG for 10 hours as in (Capowski et al, 2001
). Proteins were purified from inclusion bodies as described in (Capowski et al, 2001
). Eluted proteins were dialyzed against 1X PBS, 10% glycerol and concentrated with Centricon-10 (TAT-GFP) or Centricon-30 (TAT-RCK) centrifugal concentrators (Amicon) according to manufacturer's instructions.
2.10. TAT-fusion protein transduction into SH-SY5Y cells
SH-SY5Y cells were seeded at a density of 1.5×105 cells/well and used for TAT-fusion protein transduction when cell density reached approximately 80%. TAT-fusion protein was added into the media to a final concentration of 50 nM. Every 6 hours, media was aspirated and replaced with fresh media, to which fresh TAT-fusion protein was added.
2.11. cDNA transfection of SH-SY5Y cells
SH-SY5Y cells were seeded onto Human Placental Collagen Type VI coated coverslips in 12-well plates, at a density of 1.5×105 cells/well and cultured for 48 hours prior to transfection. Transfection was performed with 0.4 μg of pIRES-RCK-hrGFP II or pIRES-hrGFP II, or 1 μg of CMV-eGFP-APP 3′-UTR or CMV-eGFP-APP52sce 3′-UTR, and Lipofectamine® (Invitrogen) according to the manufacturer's instructions.
2.12. Real Time PCR
PCR primers were designed with Primer Express software from Applied Biosystems (Foster City, CA) and BLAST homology searches of the amplicons revealed the primers to be gene-specific. PCR reactions were optimized for primer and template concentrations and contained 200 nM APP ORF primers (accession #X06989; ORF FWD: 1479 – tggccctggagaactacatca – 1499; ORF REV: 1565 – cgcggacatacttctttagcatatt – 1541), 50 nM eGFP primers (accession #AJ890283 FWD: 625 - agcaaagaccccaacgagaa – 644; REV: 684 – ggcggcggtcacgaa – 660), 200nM α-tubulin primers (accession #NM_006000 FWD:666 – tggtggacaacgaagcaatc – 685; REV: 726 – gttgggcgctcgatgtct – 709) or 100 nM S26 primers (accession #NM_001029; forward: 406 – cgcagcagtcagggacattt – 425, reverse: 474 – ttcacatacagcttgggaagc – 454), 10.5 μl 1:5 diluted RT reaction and 12.5 SYBR green PCR mix in a 25 μl reaction volume. The cycle conditions were: 2 min at 50 °C, 10 min at 95 °C, (40 cycles: 15 sec at 95 °C, 1 min at 60 °C), followed by a dissociation stage for 15 sec at 95 °C, 1 min at 60 °C, 15 sec at 95 °C. The average PCR efficiencies for all three sets of primers were 100 ± 5%.
2.13. Confocal microscopy and APP/RCK analysis by immunohistochemistry (IHC)
Immunohistochemistry and confocal microscopy were performed essentially as described in (Westmark et al, 2006
). Briefly, SH-SY5Y cells were fixed to Collagen VI-coated coverslips with 4% paraformaldehyde and permeabilized with 100% methanol. Cells were stained with 0.5 μg/ml anti-Amyloid Precursor Protein polyclonal antibody (Zymed) and 3 μg/ml anti-rck/p54 monoclonal antibody (Amaxa) in 1X DPBS/1% FBS, stained with Rhodamine Red™-x goat anti-mouse IgG (H+L) and AlexaFluor® 633-x goat anti-rabbit IgG (H+L) (Invitrogen) diluted 1:500 in 1X DPBS/1% FBS, and mounted on microscope slides. GFP and rhodamine were imaged as in (Westmark et al, 2006
); AlexaFluor® 633 was imaged at 633nm (excitation) and 650nm (emission). Image acquisition and analysis of relative red/green/blue fluorescence intensities with ImageJ software were carried out as described in (Westmark et al, 2006
). Approximately 50 cells were quantitated for each data set.