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Although the knowledge that nuclear and cytoplasmic proteins are modified with N-acetylglucosamine …

Biology Articles » Biochemistry » Enzymology » Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development » Materials and Methods

Materials and Methods
- Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development

Mammalian expression plasmids pCMX–mSin3A, pCMX–Flag–SMRT,and pCMX–Flag–N-CoR were generously provided byR. Evans. pcDNA3–Flag–HDAC1 was kindly providedby E. Seto. pcDNA3–OGT and G5–luc reporter constructswere described previously (Yang et al., 2001Go). pcDNA3.1–HA3–OGTand Sp1–G5–luc reporter constructs were describedpreviously (Yang et al., 2002Go). pcDNA3.1–NCOAT was providedby G. Hart. 3xFlag–TEV–2xMyc tag was a kind giftfrom D. Crawford. The 3xFlag–TEV–2xMyc tag was movedto pcDNA3.1–NCOAT to create the Flag–NCOAT expressionvector. For protein expression in mammalian cells, the indicatedmutants were subcloned into pcDNA3.1 (Invitrogen, Carlsbad,CA), whereas pGEX-2T (Amersham Biosciences Corp., Piscataway,NJ) was used for GST fusion protein expression in E. coli.

Cell culture and transient transfection
Cells were maintained under standard conditions. MCF7 cellswere estrogen deprived in Phenol Red-free media supplementedwith 10% charcoal-stripped fetal bovine serum (FBS). Transienttransfection of HepG2 cells was performed by electroporation(Yang et al., 2001Go). Luciferase activities were assayed 18–24h later. Transfection efficiencies were normalized using a cotransfectedß-galactosidase plasmid. Each transfection was donein triplicate and repeated 3–5 times. Transient transfectionof BSC40 cells was by electroporation, followed by infectionwith vTF7 (Moss et al., 1990Go) to induce protein expression.Cells were harvested 12–18 h later for protein purification.MCF7 cells were transfected with Lipofectamine 2000 (Invitrogen).

GST pull-down assays and coimmunoprecipitation assay
GST pull-downs and coimmunoprecipitations were performed aspreviously described (Yang et al., 2002Go). Precipitation wascarried out using ANTI-FLAG M2 affinity Gel (Sigma A2220, St.Louis, MO) or antibody followed by 1:1 protein A: protein Gbeads (Amersham). Antibodies used were {alpha}-HA (Roche 12CA5, Indianapolis,IN), {alpha}-mSin3A (Santa Cruz K-20, Santa Cruz, CA), {alpha}-GST (SigmaG1160), {alpha}-Flag (Sigma F3165), {alpha}-N-CoR (Santa Cruz N-19), {alpha}-OGT(provided by G. W. Hart; Kreppel et al., 1997Go), and {alpha}-NCOAT (raisedin rabbits against the intact protein and affinity purifiedas previously described; Shin et al., 1992Go). Phosphatase cocktailcontained equal units of protein phosphatase 1-{alpha} (Sigma), proteinphosphatase 1-{gamma} (Calbiochem, SanDiego, CA), and protein phosphatase2A (Upstate, Charlottesville, VA). Treatment consisted of a30 min incubation at 30°C in low stringency binding buffer(LSB).

Chromatin immunoprecipitation
MCF-7 cells transfected using Lipofectamine 2000 (Invitrogen)were treated with 100 nM 17ß-estradiol (Sigma) for2 h. Cells were processed as previously described (Yang et al.,2002Go). Where indicated, DNA quantity was determined by Southernblot analysis. Linearity of polymerase chain reaction (PCR)amplification for the indicated genes was demonstrated by serial3-fold dilutions of the input DNA.

Northern/Southern blot analysis
Northern and Southern blot analyses were performed as previouslydescribed (Roh et al., 2001Go). Hybond-N+ nylon membrane (AmershamPharmacia Biotech) was used as described in the manufacturer’sprotocol. The membranes were probed using 32P-labeled probesas in the figure.

ß-Galactosidase staining of mammary tissue
Confirmation of rtTA transgene expression was carried out bybreeding the three transgenic founder lines with a tetracycline-responsivelacZ transgenic mouse line (tetOLacZ) obtained from Dr J. Segre.Mating partners, each containing the transgenes for MMTV–rtTAand tetOLacZ gave rise to mice consisting of a mixture of genotypes.Postpubertal mice containing both transgenes were fed Diet-Dox(2 mg/mL Dox [Sigma-Aldrich] containing 1 mL "Sweet’NLow" per 100 mL drinking water) for 3 days at which point, theywere sacrificed and the mammary tissue was collected and processedby established methods.

Mammary gland whole mounts and tissue section immunofluorescence
Inguinal and thoracic mammary glands were dissected from miceat 8 weeks of age and prepared as previously described (Boweet al., 2002Go). Immunostaining was performed using rabbit anticathepsinD 1:500 (Santa Cruz Biotechnology) followed by chicken antirabbitAlexa 594 at 1:1000 (Molecular Probes, Carlsbad, CA). Nucleiwere counterstained with DAPI (Sigma) and mounted using Vectasheild(Vector Laboratories, Burlingame, CA). Normal goat IgG (SantaCruz) was used as a negative control (data not shown). Sectionswere analyzed using fluorescence Leica (Nussloch, Germany) DMRBmicroscope with image captured by a Leica DFC480 camera andstandard filter set.

Generation of TetRE–NCOAT(GK) transgenic mice
The TetRE–NCOAT(GK) transgenic mouse line was constructedas a chimera of the "short GK" NCOAT sequence (350–1110)inserted into the corresponding mouse sequence (350–1400)using unique BlpI–NcoI restriction sites. The cDNA forthe mouse brain NCOAT was cloned as described above for theGK rat. The full-length 2.75 bp PCR product was cloned intothe TetRE–SV40 transgene cassette used previously (Rohet al., 2001Go) into XbaI sites. A 5'-UTR sequence was synthesizedfrom a mouse brain cDNA library using the First Choice 5' RLM-RACEkit from Ambion (Austin, TX). The 5' oligo included a BamHIrestriction site in the "nested" oligo and the 3' antisenseoligo was designed to anneal to mNCOAT, 350 bases downstreamfrom the ATG; 5'-CATGGTACCTCGTGCAGCAGAGATCAGAGTC-3'. The resulting550-bp PCR fragment was digested with BamHI/BlpI and insertedbetween these same sites in the TetRE–mNCOAT transgene,providing a 5'UTR of about 100 bp. The 760-bp GK sequence wasinserted into the mouse sequence to complete the chimeric transgenewhich was then excised from the plasmid with SphI–ClaIand microinjected into BL/6xSJL mouse zygotes as described above.Identification of four founders out of 22 mice was performedas above using oligonucleotides to TetRE (Roh et al., 2001Go)and the reverse primer for NCOAT (5'-AAG TTG CTC AGC TTC TTCCAC TG-3'), resulting in a 460-bp PCR fragment. Northern blotanalysis showed good expression of the NCOAT(GK) transgene transcript.

Generation of MMTV–rtTA transgenic mice
The MMTV-ß-globin transgene cassette, originally providedto us by Dr H.L. Moses (Pierce et al., 1993Go), has been describedearlier in the construction of the MMTV–mEGFtr transgene(Xie et al., 1997Go). The 1 kb rtTA sequence (Clontech, Palo Alto,CA) was inserted into the EcoRI site of ß-globin exon3, replacing EGFtr. The complete 3.7 kb XhoI transgene fragmentwas purified by electrophoresis on 1% agarose, elution, andextraction using the QIAquick Gel Extraction Kit (Qiagen, Valencia,CA). The transgene DNA was microinjected into one-cell BL/6xSJLmouse zygotes at a concentration of 2 ng/µL at the Universityof Alabama at Birmingham Transgenic animal facility under thedirection of Dr Carl Pinkert. Twenty-nine potential transgenicfounders were derived and their tail DNA was isolated as previouslydescribed (McAndrew et al., 1995Go). Gene-positive animals weredetermined by PCR analysis using oligonucleotides designed toanneal to the MMTV sense sequence, 5'-TGCAACAGTCCTAACATTCA-3',and rtTA antisense sequence, 5'-TGAATGTTAGGACTGTTGCA-3'. A DNAthermal cycler (PE Applied Biosystems GeneAmp pcr System 9700)was used under the following conditions for 36 cycles: 94°Cfor 30 sec, 55°C for 45 sec, and 72°C for 45 sec. Threegene-positive founder mice were identified as a 1345 bp PCRproduct on 1% agarose. Expression of rtTA was determined bynorthern blot analysis of isolated MMTV-targeted tissues. Twofounder lines (4–1 and 6–2) gave appropriately sizedmessage (1.7–1.8 kb) corresponding to the ß-globinand the rtTA sequences.

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