Reagents and Antibodies
Protein A-Sepharose was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Geldanamycin (GA; 345805), MG132 (474790), ALLN (208719), and radicicol (553400) were purchased from Calbiochem (La Jolla, CA). Anti-rabbit Hsp90 (sc-7947), anti-mouse Hsp70 (sc-24), anti-rabbit GST (sc-459), anti-mouse GFP (sc-9996), anti-mouse HA (sc-7392), anti-mouse phospho-ERK (sc-7383), anti-mouse actin (sc-8432), and anti-mouse ubiquitin (sc-8017) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse FLAG (F-3165) was purchased from Sigma (St. Louis, MO). Anti-human rpS3 was purchased from BioInstitute (Korea University, Seoul, South Korea). ECL reagents were purchased from Pierce (34078; Rockford, IL). The glutathione-Sepharose 4B was obtained from Amersham Pharmacia Biotech (17–0756-01; Piscataway, NJ).
Cell Cultures and Inhibitor Treatment
Human embryonic kidney 293T (HEK293T) cells were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and grown at 37°C in a humidified atmosphere of 5% CO2. To explore the function of Hsp90 in the system, HEK293T or HT1080 cells were treated with GA, a specific inhibitor of Hsp90 function, at a final concentration of 1.5 μM for the indicated amounts of time.
Transfection and Generation of a Stable Cell Line Expressing FLAG-tagged rpS3
HEK293T cells were transfected with a variety of plasmids using Lipofectamine (Invitrogen) according to the instructions of the manufacturer. The expression of appropriate proteins was confirmed by Western blotting. The HT1080 cell line, which stably expressed FLAG-tagged rpS3, was generated using Lipofectamine reagent (Invitrogen). The pcDNA3-FLAG-rpS3 plasmid was transfected into HT1080 cells; clones were selected in DMEM with 5% FBS and 600 μg/ml G418 (Calbiochem; 345810). Individual clones were maintained in the same medium, which contained 40 μg/ml G418.
In Vitro Pulldown Assay Using Glutathione-S-Transferase
The complete coding region of rpS3 was inserted into pGEX-5X-1 (Amersham Bioscience). For the glutathione-S-transferase (GST) pulldown assay, full-length rpS3 fused to GST (GST-rpS3) or GST alone was expressed in Escherichia coli BL21, which was then purified on glutathione (GSH)-Sepharose 4B beads (Amersham Pharmacia). HEK293T cells in 60-mm dishes (5 × 106 cells/dish) were transfected with GFP-tagged Hsp90 using Lipofectamine. At 24 h posttransfection, cells were rinsed three times with 1 ml of ice-cold phosphate-buffered saline (PBS) and sonicated in 1 ml lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM NaF, and 1 mM Na3VO4, containing protease inhibitors). Cell lysates were spun at 12,000 × g for 10 min at 4°C to remove debris. Supernatants were incubated for 12 h at 4°C with GST or GST-rpS3 bound to beads. The beads were then washed three times in lysis buffer. Proteins were boiled in 2× SDS sample buffer, subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted using anti-GFP antibody.
Western Blot Analysis
Cells were washed with cold PBS (pH 7.4) and trypsinized. Cell suspensions were sonicated on ice, and protein concentrations were determined using the Bradford reagent (Bio-Rad, Richmond, CA). Total proteins (80 μg) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (45 mA overnight). Membranes were blocked with 5% nonfat dry milk for 1 h at 4°C. Blots were incubated with primary antibody (1:1000 dilution) in a blocking solution for 1 h at 4°C. Membranes were rinsed twice with TBST (1% Tween-20 in Tris-buffered saline, pH 7.4) and incubated with secondary antibody conjugated to HRP (1:2000) in a blocking solution for 30 min at 4°C. The bound complex was visualized using the chemiluminescent Super Signal kit (Pierce).
Cells were harvested on ice in the after lysis buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, and protease inhibitors (2 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A). Proteins were extracted by ultrasonication and centrifuged (16,000 × g for 20 min at 4°C); the supernatants were then collected and incubated with 2 μg of primary antibodies for 4 h at 4°C. The immunoprecipitates were harvested using protein A-Agarose beads. After extensive washing, immunoprecipitates were eluted by 5-min boiling of the beads in 2× SDS-PAGE sample buffer. The samples were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and characterized by Western blotting with appropriate antibodies.
293T cells were plated on poly-d-lysine-coated multiwell chamber slides (Becton Dickinson, Lincoln Park, NJ) and incubated for 1 d. The cells were then fixed with 3.7% paraformaldehyde in PBS, quenched with 50 mM NH4Cl in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Next, the cells were incubated with rabbit anti-rpS3 or mouse anti-Hsp90 antibodies for 1 h at room temperature. The Texas Red (red) goat anti-rabbit antibody and FITC (green) goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for rpS3 and Hsp90, respectively. Stained cells were analyzed under a confocal microscope (Bio-Rad).
Recombinant rpS3 cDNA was transfected with Lipofectamine according to the instructions of the manufacturer. After 24 h, transfected cells were supplied with fresh media containing 1.5 μM GA or 20 μM radicicol. The cells were incubated in the presence or absence of Hsp90 inhibitors for the indicated amounts of time. Cell harvesting and lysis were performed as described in immunoprecipitation method. In brief, immunoprecipitations were performed using equal amounts of whole cell extracts. Lysates (minimum of 400 μg for each condition) were incubated with 2 μg of antibodies (mouse anti-FLAG or anti-GFP), and immune complexes were bound on protein A-Sepharose beads for 6 h at 4°C with rotation. The beads were washed three times with ubiquitination buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, and 1% SDS) and bound proteins were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis.
For sucrose density gradient fractionation of ribosomes, cells were harvested after adding cycloheximide (100 μg/ml) to the medium for 15 min. The cells were then homogenized using 0.3% NP-40 lysis buffer containing 30 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 100 mM KCl, 50 μg/ml cycloheximide, 30 U/ml RNasin inhibitor, 1 mM dithiothreitol, 1 mM PMSF, 1 mM pepstatin, 1 mM aprotinin, and 1 mM leupeptin for 5 min at 4°C. The lysates were centrifuged at 12,000 × g at 4°C for 8 min, and the supernatants were collected and measured at OD260 to determine the RNA concentration. The lysates containing 800 μg of RNA were loaded to a 10–50% sucrose gradient solution containing 30 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 100 mM KCl. The gradients were centrifuged at 37,000 rpm for 2 h at 4°C using a Beckman SW40Ti rotor. One-hundred twenty fractions were collected from each tube, measured for absorbance at 254 nm using spectroscopy, and analyzed by Western blotting.
For the collection of ribosomal and nonribosomal fractions, the lysates were centrifuged at 10,000 × g at 4°C for 15 min in order to remove the mitochondria and cell debris. The supernatant was layered over a sucrose (20% wt/vol) cushion containing cycloheximide and centrifuged at 149,000 × g for 2 h. The pellet containing ribosomes and the upper and lower pellets of the nonribosomal supernatants were collected. The ribosomal pellets were resuspended in the lysis buffer, after which immunoblotting was performed.
Purification of Nucleoli
Nucleoli were isolated from 293T cells according to the method of Padilla et al.
), with slight modifications. 293T cells were harvested and washed three times with 1 ml of ice-cold PBS, suspended in 20 ml of RSB buffer (10 mM Tris·HCl, pH 7.5/10 mM NaCl/1.5 mM MgCl2
), incubated on ice for 30 min, and centrifuged at 500 × g
for 5 min at 4°C. Cells were suspended in 0.6 ml of RSB buffer containing 0.5% (wt/vol) NP-40 (IGEPAL CA-630, Sigma) and were then homogenized (Fisher Scientific, Springfield, NJ; 10 strokes with a tight pestle). The homogenate was centrifuged at 1200 × g
for 5 min at 4°C, and the nuclear pellet (dispersed in 10 volumes of 0.25 M sucrose containing 3.3 mM CaCl2
) was layered over 1 ml of 0.88 M sucrose. After centrifugation at 1200 × g
for 10 min at 4°C, the pelleted nuclei were dispersed in 10 volumes of 0.34 M sucrose and sonified. Sonified nuclei were layered over 1 ml of 0.88 M sucrose and centrifuged at 2000 × g
for 20 min at 4°C. The nucleolar pellet was washed twice with 0.34 M sucrose and was stored at -80°C in 0.1 ml of the same solution. Proteins from each fraction were separated by SDS-PAGE in 10% gels, transferred to nitrocellulose, reacted with the indicated antibodies, and detected using chemiluminescence.
Subconfluent monolayers of HEK293T cells were treated with GA for 12 h in complete media and starved in methionine-free DMEM for 1 h before labeling with the 40 μCi/ml [35S]methionine/cysteine mixture for several minutes. To measure the translation activity of the ribosomes, labeled cells were harvested and lysed. Equal amounts of proteins were separated by SDS-PAGE in 10% gels. After the gel was dried, labeled proteins were visualized with a BAS2500 imaging analyzer (Fujifilm, Tokyo, Japan).