5. MATERIAL AND METHODS
5.6. Xenopus egg extracts
UbcX dissociates XErp1 from the APC/C Material and Methods
61 was performed using specific primary antibodies (see Table 4.3), commercially available HRP-‐conjugated secondary antibodies (horse radish peroxidase; Bio-‐
Rad and Dianova) and ECL (100 mM Tris-‐HCl pH 8.5, 1.25 mM luminol, 225 μM coumaric acid, 0.015% v/v H2O2) or ECL plus (Lumigen TMA-‐6, GE Healthcare) reagents.
5.6. Xenopus egg extracts
5.6.1. Xenopus CSF egg extract preparation
Three days before extract preparation, female Xenopus laevis were primed by injecting 50 U PMSG (gonadotropin, pregnant mare serum in H2O, Merck) into the dorsal lymph sack. One day before extract preparation, frogs were induced to ovulate by the injection of 800 U HCG (human chorionic gonadotropin in H2O, Sigma) into the dorsal lymph sack. Frogs were put into tanks filled with 2L of 1x MMR [0.1 M NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES pH 7.8] for ovulation. Laid eggs of sufficient quality were collected, washed with 2L 1x MMR and treated with dejellying solution for up to seven minutes. Eggs were then washed with 1L CSF-‐XB [2 mM MgCl2, 0.1 mM CaCl2, 100 mM KCl, 5 mM EGTA, 50 mM sucrose, 10 mM HEPES pH 7.7]. Activated and lysed eggs were removed and the eggs were transferred into centrifugation tubes pre-‐filled with 1 ml CSF-‐XB containing 0,1 mg/ml cytochalasin B (Sigma). The eggs were compacted by a two-‐step centrifugation for 1 minute at 160xg and another minute at 650xg in a swing out rotor. Excess buffer was removed from the tube after centrifugation leaving the compacted, eggs behind. In a final centrifugation step the eggs were then lysed by centrifugation for 10 minutes at 17.000xg at 4 °C. The cytoplasmic layer was then isolated with a 1 ml syringe by puncturing the tube at the bottom of this layer. The extract was supplemented cytochalasin B (10µg/ml, Sigma) and stored on ice for further use.
UbcX dissociates XErp1 from the APC/C Material and Methods
5.6.2. Extract manipulations
All incubations were done at 20°C. When indicated, CaCl2 was added to 600 µM, cycloheximide to 350 µM, MG262 to 100 µM, nocodazole to 33 µM and Ocadaic acid to 2 µM. “High sperm” and “low sperm” extract contained 11,5x106 and 1x106, respectively, sperm nuclei per ml extract. In general, 6xhis-‐
UbcH10wt, 6xhis-‐UbcH10ci were added to a final concentration of 12 µM, 6xhis-‐
UbcXwt or 6xhis-‐UbcXci to 6 µM (unless explicitly mentioned), MBP-‐XErp1 CT (aa 374-‐651) to 200nM, Ubiquitin-‐aldehyde (Boston Biochemicals) to 6 µM and a corresponding volume of dialysis buffer was used as buffer control. To dephosphorylate extract proteins, samples were diluted with 9 volumes of 1x CIP-‐buffer (NEB) containing 10 units CIP (NEB) and incubated for 30 min at 25°C. For glycerol-‐gradient centrifugation, extracts were cleared by centrifugation at 186.000xg and resolved on 10–40% glycerol gradient by centrifugation at 150.000xg. 0.5 ml fractions were collected and analyzed by SDS-‐PAGE and immunoblotting.
5.6.3. Immunodepletion
For the depletion of 100µl egg extract, 3x10 µg Cdc20, Cdc27 or approximately 3x15 µg βTRCP antibodies as well as the corresponding control antibodies were coupled to 3 x 25 µl protein A Dynabeads (Pierce) in 100µl PBS-‐Tx [1xPBS, 0,1%TritonX100] for 1h at room temperature rotating on a wheel. The antibody beads were washed with 500µl PBS-‐Tx, 500µl PBS-‐Tx containing 500mM NaCl and 3x with 500µl CSF-‐XB [2 mM MgCl2, 0.1 mM CaCl2, 100 mM KCl, 5 mM EGTA, 50 mM sucrose, 10 mM HEPES pH 7.7]. Every time, the beads were collected by putting them in the testube on a magnet; only after the last washing step, the beads were additionally centrifuged for 10 s in a nanofuge and all residual buffer was removed carefully. Next, the extract was incubated with the antibody beads in three rounds for 30 min on ice. Every time the beads were removed from the extract by incubating the test tube for 10 min
UbcX dissociates XErp1 from the APC/C Material and Methods
63 on ice on the magnet, and the extract was added to new antibody-‐beads. After the third round, the beads were carefully removed and the depleted extract was used for further experiments.
5.6.4. Immunoprecipitation
For Cdc27 immunoprecipitation assays, the 250 µl extracts were treated as indicated, diluted 1:1 with CSF-‐XB buffer and incubated with 5 µg anti-‐Cdc27 antibodies coupled to 30 µl protein A agarose beads. The reaction was incubated for 4 h at 4°C rotating on a wheel. The beads were retrieved by centrifugation, washed three times with 1 ml CSF-‐XB and resuspended in 3x laemmli buffer for westernblot analysis. To quantify immunoprecipitated Cdc27 and XErp1, immunoblots were visualized with ECL reagent. Signal intensities were quantified using Image J software. Anti-‐Cdc27 immunoprecipitates were used as loading controls and the relative amounts of copurified XErp1 were calculated.
XErp1 and Cdc20 immunoprecipitation assays were performed like described for Cdc27 immunoprecipitations, except 125 µM N-‐ethylmaleimide was added when the extracts were diluted with CSF-‐XB.
For the purification of his-‐tagged ubiquitin conjugates a protocol described in (Glockzin et al., 2003) was modified with the help of Alejandro Rojas. Briefly, to CSF extract 12,5 µM 6xhis-‐Ubiquitin or 6xhis-‐UbiquitinDGG was added and incubated with 6xhis-‐UbcXwt, 6xhis-‐UbcXci or buffer at 20°C for 30’. The extract was denatured at 4°C by adding 9 volumes of denaturation buffer (6M guanidinium-‐HCl, 100 mM Na2HPO4/NaH2PO4 pH 8.0, 10 mM imidazole, 10 mM β-‐mercaptoethanol and complete protease inhibitors (Roche)), cleared by centrifugation and incubated with Ni2+-‐NTA beads (Quiagen). The beads were washed, the bound proteins were re-‐natured, eluted into 3x laemmli buffer containing 200mM Imidazole and analyzed by Western blotting.
UbcX dissociates XErp1 from the APC/C Material and Methods
5.6.5. In vitro ubiquitylation assays
APC/C was immunoprecipitated from 2 ml CSF extract using 25 µg Cdc27 antibodies immobilized on protein G dynabeads (Invitrogen). Assays were done as described previously (Schmidt et al., 2005), except that no exogenous Cdc20 or XErp1 was added: The beads were washed once in QA [100 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM Tris pH 7,5] and twice in CSF-‐XB and divided into 10. A mix containing E1 (110 nM; Boston Biochemicals), Ubiquitin (1,25 mg/ml;
Boston Biochemicals) and an energy regeneration system [1 mM ATP, 20 mM creatinephosphate, 150 µg /ml creatine-‐phospho-‐kinase (Sigma)] and UbcX (12,5 µM) in CSF-‐XB buffer was added and the reaction was started by the addition of UbcX. For Cdc20 ubiquitylation assays, an equal volume of 3x laemmli buffer was added to the reaction at the indicated time points. To assay XErp1 ubiquitylation and release from the APC/C, the beads were separated from the supernatant on a magnet and the supernatant was added to an equal amount of 3x laemmli buffer. Samples were directly processed and analyzed by WB.