NY). Beta-actin and a-tubulin antibodies, cycloheximide and MG132 were from Sigma (St. Louis, MO). Construction of vectors nCLU was constructed by polymerase chain reaction (PCR) and cloned into pcDNA3.0 vector (with HA tag) or pcDNA3.1 vector (with Myc tag). Deletion forms of VHL were cloned into pCDNA3.1 vector or into pET-28a Vector (with His epitope). R-VHL was constructed through inserting VHL into pDsRed- Monomer-N1 vector between Xho I and Hind III. Glutathione S- transferase (GST)-nCLU fusion proteins were constructed by inserting PCR-generated DNA fragments encoding nCLU into pGEX4T1. E. coli BL21-Gold(DE3)pLysS cells were transformed with pGEX4T1 or pET28a expression vectors and treated with 0.1 mmol/L of isopropyl-D-thiogalactoside for 4 h. Ubiquitin was constructed by PCR and various mutated ubiquitin constructs were generated by site-directed mutagenesis. The diagram of ubiquitin mutants is as follows. 6 11 27 29 33 48 63 Ub ----K-----K----K----K------K------K-----K--- Ub(K48) ----R-----R----R----R-----R------K-----R--- Ub(K63) ----R-----R----R----R-----R------R-----K--- Ub(K63R) ----K-----K----K----K------K------K-----R--- Ub(K48R) ----K-----K----K----K------K------R-----K--- Immuno-staining of cells The cells, grown on glass coverslip were washed and fixed with 4% formaldehyde for 20 minutes, followed by permeabilization with 0.1% Triton X-100. After wash, the coverslips were blocked in 3% BSA for 1 h, followed by the incubation with primary antibody at 4uC overnight. After wash, the coverslips were incubated with fluorescence dye-conjugated secondary antibody for 30 min. Finally, the cells were stained with DAPI in the dark for 3 min. The stained cells were mounted and observed in A Zeiss LSM 510 META (Axiovert 200) microscope. Nuclear protein extraction Cells were washed and suspended in 100 mL of buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM EGTA, 0.1 mM EDTA, 0.5 mM phenyl- methylsulfonyl fluoride, and 0.2 mM phenylmethylsulfonyl fluo- ride, 0.5% Nonidet P-40). The cells were lysed, followed by centrifugation at 17006 g for 10 min. After separation of the cytoplasmic fraction, nuclei were washed once with Buffer A and resuspended in ice-cold buffer B (10 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2 and 0.4 mM NaCl, 1 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride). After 1 h incubation on ice and a centrifuge to clear the cellular debris, the nuclear extracts were collected and used for immunoblotting. Transient transfection Transient transfection of cells was performed using LipofectA- MINE-2000 or LipofectAMINE-PLUS (Invitrogene) as per the manufacturer’s instructions. Immunoprecipitation and immunoblotting Immunoblotting and immunoprecipitation was performed as described [24]. Briefly, 500 mg of cell lysates were incubated with 1 mg of primary antibody at 4uC for 3 h. Then, 20 mL of protein A/G PLUS agarose beads was added and the incubation continued overnight. The beads were washed and boiled in SDS-PAGE loading buffer for 5 minutes before electrophoresis. pVHL Mediates Ubiquitination of nCLU GST pull-down assay GST pull-down assay was performed as described [24]. Bacterial cells were lysed using the following buffer: 20 mmol/L Tris-Cl, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% NP40, pH 7.5. To determine the interaction between nCLU and pVHL, bacterial lysates containing GST-nCLU were incubated with glutathione-Sepharose 4B beads at 4uC for 1 h. The beads were washed and incubated with bacterial cell lysates containing His- pVHL, allowing the interaction between GST-nCLU and pVHL. After washing, GST-nCLU and the bound pVHL were eluted from the beads and subjected to electrophoresis. In vitro ubiquitination assay In vitro ubiquitination assay was performed as described [25] and the Reaction II reticulocyte conjugation kit (Bostonbiochem, MA) was employed in this assay. To isolate the pVHL complex, Myc-VHL was transiently expressed in 293T cells and four hundred micrograms of the cellular proteins were immunoprecip- itated with 1 mg of Myc antibody at 4uC for 3 h. Twenty microliters of protein A/G PLUS agarose beads were added and the incubation continued at 4uC overnight. The beads were washed for four times with RIPA buffer, followed by two washes with buffer containing 50 mM Tris-HCl and 3 mM DTT, pH 8.0. The agarose beads that contained purified pVHL complex were suspended in a final reaction volume of 50 mL containing ATP-regenerating buffer, 5 mg/mL ubiquitin, 1 mg/mL GST-nCLU, 100 ng/mL ubiquitin aldehyde, MG132 (5 mM), and 16 mL of purified rabbit reticulocyte fraction II, and incubated at 37uC for 2 h. After reaction, the total proteins generated in the ubiquitination reaction were analyzed by western-blot. Statistical analysis The data represent the mean 6 SD from three independent experiments except where indicated. Statistical analysis was performed using the unpaired two-tailed Student’s t-test. The difference is considered significant when a P value is less than 0.05. Results pVHL interacts with nCLU pVHL is an adaptor protein and binds to many proteins. We determined whether pVHL had an interaction with sCLU and nCLU. 293T cells were transiently transfected with HA-VHL plus Myc-sCLU or HA-VHL plus Myc-nCLU plasmids. Immunopre- cipitation was performed and the results indicated that HA-pVHL co-immunoprecipitated with Myc-nCLU (Fig. 1A, Fig. S1A) but not Myc-sCLU (Fig. 1A), indicating that pVHL had an interaction with nCLU. We repeated the experiment in 293T cells to confirm the interaction between pVHL and nCLU. The cells were transfected with Myc-VHL and HA-nCLU vectors. The results showed that Myc-pVHL and HA-nCLU also co-immunoprecip- itated (Fig. 1B, Fig. S1B). A direct interaction between pVHL and nCLU was ascertained by GST-pull down assays (Fig. 1C). These results suggest that pVHL binds to nCLU directly. We determined the domain of pVHL that nCLU might bind to. pVHL contains two functional sub-domains termed a and b, and the b domain (residues 63–155) functions for recognition of substrate. GST-pull down assay was performed using pVHL fragments covering different regions. VHL encodes wild-type pVHL, VHL(1-155) encodes pVHL without a domain, and VHL(D63-155) encodes pVHL without b domain. The results showed that pVHL and pVHL(1-155) bound to nCLU (Fig. 1D). However, pVHL(D63-155) had no interaction with nCLU. The PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35848