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A chemical cross-linking method for the analysis of binding partners of heat shock protein-90 in intact cells
 
Shaoming Song1, Sutapa Kole2, and Michel Bernier1
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Protein cross-linking in intact cells

The human HepG2 hepatocarcinoma and 1321N1 astrocytoma cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in α-MEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM pyruvate, 50 U/mL penicillin and 50 µg/mL streptomycin. The human melanoma cell line lacking FLNa (M2 cells) and the isogenic cell line stably transfected with a full-length FLNa construct (A7 cells) were previously described (21). All cell lines were cultured at 37°C in a humidified incubator with 5% CO2, and the medium was replaced every 2–3 days. Peripheral blood mononuclear cells (PBMC) collected from healthy donors (who provided informed consent) were isolated by Ficoll-Hypaque density gradient centrifugation. Cells were cultured in RPMI 1640 with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine for 16 h.

Before each experiment, cells were incubated with serum-free α-MEM:Ham's F-12 (1:1) for 4 h, washed twice in phosphate-buffered saline (PBS), and then incubated either with vehicle or various protein cross-linkers (1 mM) for 30 min at 20°C in PBS. The protein cross-linking reaction was quenched by the addition of an excess of glycine (10 mM). Cells were rinsed and immediately processed as indicated below. To assess the role of Hsp90 binding drugs, serum-starved cells were pretreated with vehicle (dimethyl sulfoxide; DMSO), 17-AAG (5 µM), DMAG (5 µM), or novobiocin (0.2–0.6 mM) for 1 h before EGS cross-linking reaction. Based on the nature of the experiments, the use of serum starvation prior to cross-linking reaction in PBS was to eliminate cross-reaction of the EGS with serum proteins and free amino acids present in the culture medium.

Isolation of cellular organelles

Cell mitochondria were isolated according to the manufacturer's protocol (MitoScience, Eugene, OR, USA). In brief, cells were subjected to three freeze-thaw cycles followed by a two-step homogenization procedure using Dounce homogenizer with Teflon pestle. The clarified crude homogenates were centrifuged at 12,000× g for 10 min at 4°C, and the pelleted mitochondria were resuspended, aliquoted, and stored at -80°C. The postmitochondria supernatant was centrifuged at 100,000× g for 1 h at 4°C to collect the crude membrane fraction and cytosol; both of which were aliquoted and stored at -80°C. Nuclear extracts from HepG2 cells were prepared using the NE-PER extraction kit (Thermo Scientific Pierce).

Plasmid construction

The cDNA encoding the human Hsp90α (HSP90AA1) with a N-terminal 94 amino acid truncation (ΔNT-Hsp90α; GenBank/EBI Data Bank accession no. BC023006) was purchased from ATCC. ΔNT-Hsp90α ligated in pCMV-SPORT6 (Thermo Fisher Scientific, Open Biosystems) was subcloned in-frame into two mammalian expression vectors, pCMV-3Tag-1 and pCMV-3Tag-2 (Stratagene; vectors contain three copies of the FLAG-tag and Myc-tag, respectively), to generate the N-terminal FLAG-ΔNT-Hsp90α and Myc-ΔNT-Hsp90α fusion constructs. All constructs were confirmed by standard DNA sequencing.

Transient transfection experiments

HepG2 cells grown to 50% confluence on 35-mm dishes were transiently transfected with either FLAG-ΔNT-Hsp90α, Myc-ΔNT-Hsp90α, or empty vector plasmid with 2 µg DNA/dish using Lipofectamine 2000 in serum-free Opti-MEM, according to the manufacturer's instructions (Invitrogen). The cells were incubated for 48 h after transfection and then serum-starved for 4 h before performing protein cross-linking reaction.

Transfection of 21-nucleotide siRNA duplexes for targeting endogenous FLNa was carried out using Lipofectamine RNAiMAX reagent and 20 nM siRNA duplex per 35-mm plate, according to the supplier's instructions (Invitrogen). Transfected HepG2 cells (250,000 cells/mL) were assayed 3 days after reverse transfection. The sequences of FLNa siRNA used were: r(GGAAGAAGAUCCAGCAGAA)dTdT (sense) and r(UUCUGCUGG-AUCUUCUUCC)dAdC (anti-sense), whereas the negative control siRNA was the AllStars Neg. Control siRNA (Qiagen) that has no known target gene. These validated siRNAs have been shown to perform efficient knockdown with minimal off-target effects. Specific silencing of FLNa was confirmed by real-time PCR and immunoblotting.

Immunoprecipitation and Western blot analysis

Cells were lysed in radioimmunoprecipitation assay buffer (50 mM HEPES, pH 7.4, 135 mM NaCl, 1%, w/v, Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 100 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail I and II; EMD Biosciences) as detailed (21). Insoluble material was removed by centrifugation (16,000× g at 4°C for 10 min), and the protein concentration in the soluble extracts was determined with the Pierce BCA protein assay (Thermo Scientific) using BSA as standard. For immunoprecipitation experiments, aliquots of the cell lysates were precleared with protein G agarose for 1 h at 4°C, and then incubated with 2 µg monoclonal anti-Hsp90α/β antibody (clone F8; Santa Cruz Biotechnology) for 16 h at 4°C. The immune complexes were collected by addition of protein G agarose for 90 min at 4°C, washed extensively, and eluted in 1.5× Laemmli sample buffer supplemented with 7.5% 2-mercaptoethanol. In some instances, cell lysates were incubated with anti-FLAG M2 affinity gel and anti-Myc-tag antibody. Immunoprecipitates and cell lysates were subjected to SDS-PAGE under reducing conditions and then transferred to polyvinylidene difluoride membranes using iBLOT apparatus. Details of immunoblotting are described elsewhere (21).

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