Rec protein g sepharose 4b conjugate

Co-IP from rice cells and N. benthamiana cells were performed as described in Liu et al. (2014). Briefly, samples were extracted with IP buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% NP-40, 5 mM DTT, protease inhibitor cocktail Complete Mini tablets [Roche]) as previously described [10 (link)]. Relevant antibodies were added to the cell lysates (10 μg ml^-1) and MG132 (Sigma) was added at a final concentration of 50 μM to prevent protein degradation. The mixtures were kept at 4°C with gentle shaking for 30 min. The IP complex was captured by adding 20 μl ml^-1 rec-protein G-Sepharose 4B Conjugate (Invitrogen; Carlsbad, CA, USA), following by shaking at 4°C for another 1 h. The Sepharose beads were recovered by centrifugation at 1000×g for 30 sec and washing three times with cold TBS (50 mM Tris-HCl pH7.5, 150 mM NaCl). Then 20 μl of sample buffer (50 mM Tris-HCl pH6.8, 2% SDS, 6% Glycerol, 0.1M DTT, 0.02% bromophenol blue) was added into the beads. After boiling for 10 min and centrifugation, the samples were loaded onto the SDS-PAGE gels for Western blotting analysis.

For co-immunoprecipitation assays between Mdm30 and Ubp2, the MDM30 ubp2Δ shuffle strain (MCY996) was cured from the MDM30 shuffle plasmid (pRS316-MDM30) and transformed with either pTEF-MDM30-MYC or pTEF-fbox-MYC. Resulting transformants were subsequently transfected with pRS423-UBP2-6HA to yield strains that co-express HA and MYC tagged versions of Ubp2 and Mdm30 as the sole source of both proteins. These cells grown at exponential phase were lysed at 4 °C with glass beads in IP buffer (100 mM Tris, pH 7.5, 100 mM sodium chloride, 0.6% Triton X-100, 10% glycerol, supplemented with protease inhibitors (Protease Inhibitor Cocktail; Sigma-Aldrich) and 1 mM Pefabloc (Sigma-Aldrich)). Insoluble material was removed by centrifugation for 30 min at 13,000g. Aliquots of supernatants were diluted and heated in sample buffer (pre-IP lysate). The remaining supernatant was incubated for 2 h at 4 °C with protein G-Sepharose beads (rec-Protein G-Sepharose 4B Conjugate, Invitrogen) in the absence (Mock) or in the presence of anti-Myc antibodies (9E10, Invitrogen, R950-25). Beads washed with IP buffer were heated in sample buffer before resolution by SDS–PAGE and analysis by immunoblotting with indicated antibodies.

For coimmunoprecipitation assays, PC-3 cells were harvested and lysed in immunoprecipitation lysis buffer29 (link). Culture media were harvested from 48-h cell cultures in T75 flasks. Cell lysates (500 μg in 0.5 mL) were mixed with 2 μg of normal IgG or antibody to Flag (Sigma-Aldrich) or V5 (Invitrogen), then incubated for 3 h at 4 °C. Following this incubation, 100 μL of rec-Protein G-Sepharose 4B conjugate (Invitrogen) was added and incubated with mixing overnight at 4 °C. After centrifugation at 3,000 × g for 1 min, supernatants were aspirated and discarded. Pellets were washed in lysis buffer 3 times for 15 min. Sample buffer (30 μL) was added to the agarose pellets, which were then boiled for 10 min. Samples were then subjected to Western-blot analysis.