Human UBXN7 [GenBank: NM_015562] was amplified from EST IMAGE 5294894. For mammalian expression, wild-type and mutant UBXN7 variants were subcloned as BamH1/Not1 inserts into pCMV5-Flag. For bacteria expression, UBXN7 variants were subcloned into a modified pGEX6P-1 vector containing a TEV protease site and a Flag tag downstream of the GST. Human CUL2 [GenBank: NM_003591] and RAD23B [GenBank: NM_002874.3] were amplified from EST IMAGE 4104375 and 3906269, respectively, for subcloning into pCMV5-Flag as BamH1/Not1 inserts. Human FAF1 [GenBank: NM_007051.2] and PSMD4 [GenBank: NM_002810.2] were amplified from EST IMAGE 5928559 and 6285035, respectively, for subcloning into pCMV5-Flag as Sal1/Not1 inserts. To construct the baculovirus vector for dual expression of GST-CUL2 and HIS6-RBX1, human CUL2 was subcloned as a BamH1/Not1 insert into the PPH driven cassette of pFastBac-Dual-GST. RBX1 [GenBank: NM_014248.2] was amplified from EST IMAGE 3138751 adding an Nhe1 site and 6HIS tag on the 5' primer and a Kpn1 site on the 3' primer and subcloned into the PP10 driven cassette of pFastBac-Dual-GST-CUL2.
PCR reactions were carried out using KOD Hot Start DNA Polymerase (Merck Millipore, Darmstadt, Germany). All full-length PCR products were cloned into pSc-B (Agilent Technologies, Santa Clara, CA, USA) and fully sequenced prior to further subcloning. All mutations and deletions were made following the Quickchange method (Agilent Technologies), but using KOD Hot Start DNA Polymerase. DNA sequencing was performed by the Sequencing Service at the College of Life Sciences (CLS), University of Dundee.
Cell Extracts and Immunoprecipitation
For immunoprecipitation experiments, the cells were lysed in buffer A (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/KOH, pH 7.2; 5 mM Mg(OAc)2; 70 mM KOAc; 0.2% Triton X-100; 10% glycerol; 0.2 mM ethylenediaminetetraacetic acid (EDTA); protease inhibitors) and incubated with anti-UBXN7 antibodies crosslinked to Protein A-agarose or anti-Flag beads (Sigma, Saint Louis, MO, USA). For the experiment in Figure , PhosSTOP phosphatase inhibitor (Roche. Mannheim, Germany) was also added to the lysis buffer.
Total extracts were prepared using buffer B (50 mM HEPES/KOH, pH 7.2; 400 mM NaCl; 1% NP-40; 0.2 mM EDTA; 10% glycerol; protease inhibitors) to facilitate extraction of nuclear HIF1α.
Antibodies and Chemicals
The following antibodies were used for protein detection by western blotting: mouse anti-Flag M2 (Sigma), mouse anti-ubiquitin FK2 (Enzo, Farmingdale, NY, USA), mouse anti-p97 (Fitzgerald, North Acton, MA, USA), mouse anti-CUL3, mouse anti-elongin C (BD Transduction Laboratories, San Jose, CA, USA), rabbit anti-CUL4A (Cell Signalling, Danvers, MA, USA), rabbit and mouse anti-CUL1, rabbit anti-CUL2, rabbit anti-NEDD8 (Invitrogen, Camarillo, CA, USA), rabbit anti-RBX1 (Thermo, Fremont, CA, USA), rabbit anti-VHL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-HIF1α (Novus, Littleton, CO, USA), rabbit anti-UBXN7 (courtesy of Millipore, Billerica, MA, USA). MG132 (Enzo) was added at 10 μM to the tissue culture media for two hours prior to cell lysis. The Division of Signal Transduction Therapy (DSTT) at the CLS, University of Dundee synthesized MLN4924, as described previously [53
]. The cells were incubated with 1 μM MLN4924 for two hours.
Recombinant Protein Expression and Purification
The Protein Production and Assay Development Team (PPADT) at SCILLS produced the various recombinant proteins in bacteria, as follows. Expression vectors for full length, UBA- or UIM-deleted UBXN7 were transformed into BL21 DE3 cells. Overnight cultures were grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) supplemented with carbenicillin. Autoinduction medium was inoculated and the cells were left to grow at 37°C until the OD600 reached about 1.5. The temperature was then dropped to 15°C and the cells were left for about 16 hours to express the protein. The cells were collected by centrifugation and resuspended in 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.4% Triton X-100, 0.1 mM EDTA, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT) and protease inhibitors. The suspension was sonicated and the insoluble material was sedimented by centrifugation at 4°C, 28,000 g for 20 minutes. The supernatant was incubated with GSH-sepharose for one hour. The sepharose was washed four times and UBXN7 was recovered upon cleavage with TEV protease. The proteins were further purified by chromatography over a Superdex 75 column after which protein purity exceeded 90%.
The vector expressing Flag-CUL1(324-776)/GST-HA-RBX1 [33
] was also transformed into BL21 cells, but grown in LB/ampicillin and induced with 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at an OD600
of 0.7. This was then left to express at 15°C overnight and the lysate was prepared as described above.
The dual expression vector encoding GST-CUL2/HIS6-RBX1 was used to generate recombinant baculoviruses using the Bac-to-Bac system (Invitrogen) following the manufacturer's protocol. These baculoviruses were used to infect Spodoptera frugiperda 21 cells (1.5 × 106/ml) at a multiplicity of infection of 5 and the infected cells were harvested 48 hours post-infection. GST-CUL2/RBX1 was purified on GSH-Sepharose and dialysed into 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 150 mM NaCl, 270 mM sucrose, 0.03% Brij-35, 0.1% 2-mercaptoethanol, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride (PMSF).
In Vitro Binding Assays
The Flag-CUL1(324-776)/GST-HA-RBX1 complex was immobilized on GSH-sepharose (GE). For each binding assay, 10 μl beads carrying approximately 1 μg CUL1/RBX1 were incubated for 30 minutes at 30°C with a neddylation reaction mix containing NEDD8 E1 (PPADT, SCILLS), NEDD8 E2 (Ubiquigent, Dundee, UK), NEDD8 and ERS (BostonBiochem, Cambridge, MA, USA) in buffer C (50 mM HEPES/KOH, pH 7.5; 60 mM KOAc; 5 mM MgCl2; 5% glycerol, 1 mM DTT). Mock neddylation reactions were performed in parallel by omitting NEDD8 from the mixture. The beads were then washed and incubated for one hour with 3 μg of wild-type or mutant Flag-UBXN7 in buffer C without DTT and supplemented with 0.1% Triton X-100 (buffer D). After washing the beads, the bound proteins were eluted with Laemmli buffer. The binding assays were also performed using naked beads to account for non-specific UBXN7 binding to the beads.
GST was cleaved off CUL2 with PreScission protease and the resulting CUL2/RBX1 was neddylated as above. CUL2/RBX1 (1.5 μg) was pre-incubated with 1.5 μg Flag-UBXN7 (approximately 1.5 times molar excess to CUL2) for 30 minutes, followed by one hour incubation with 10 μl anti-Flag beads in buffer D. The proteins bound to beads were eluted by boiling in Laemmli buffer.
Wild-type or mutant UBXN7 (25 μg) was incubated for one hour with 10 μl NEDD8- or ubiquitin-agarose (BostonBiochem) in buffer D and the bound proteins were eluted as above.
Immunofluorescence Staining and Microscopy
For Flag-UBXN7 immunostaining, cells were grown on coverslips and fixed with ice-cold methanol for six minutes at -20°C. Cells were then blocked in 1% BSA/PBS for 30 minutes and subsequently incubated with mouse anti-Flag M2 (Sigma) antibodies in 3% BSA/PBS, for one hour at room temperature. After washing with PBS, cells were incubated with donkey anti-mouse FITC-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 45 minutes. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen). The coverslips were mounted onto glass slides using hydromount (National Diagnostics, Atlanta, GA, USA).
Images were obtained with a DeltaVision Spectris microscope (Applied Precision), using a CoolSNAP HQ camera (Roper) and a 60 × 1.4 NA objective (Olympus). The SoftWorx software (Applied Precision) was used for acquisition and deconvolution.