Pureproteome protein a g magnetic beads

Cell pellets were collected from 80% confluent wild-type AGS cells and lysed in IP lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40) supplemented with protease inhibitor (Santa Cruz). Appropriate amounts of protein samples were incubated with the corresponding antibodies (1 μg) as indicated and 10 μl of PureProteome protein A/G magnetic beads (Millipore) at 4 °C overnight. The beads were washed three times with IP-wash buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Tween 20, pH 7.4) and eluted by the IP-lysis buffer, followed by immunoblotting assay with corresponding antibodes as indicated.

PureProteome protein A/G magnetic beads (Millipore) were equilibrated with anti-K (NQTGG) antibody (UMO-1-7-7, Abcam) at a ratio of 1:2 (v/w) for 1 h at 4 °C in PBS. Saturated beads were washed three times with 200 mM triethanolamine pH 8.3. For crosslinking, 10 μl of 5 mM dimethyl pimelimidate in 200 mM triethanolamine pH 8.3 was added per microliter of slurry and incubated for 1 h at room temperature (RT). The reaction was quenched for 30 min by adding 1 M Tris-HCl pH 8 to a final concentration of 5 mM. Crosslinked beads were washed three times with ice-cold PBS and once with PBS containing 50% glycerol. The tryptic digests were resuspended in 500 μl PBS containing 50% glycerol and supplemented with crosslinked anti-K-(NQTGG) at a ratio of 1:2 (w/w). After 1 h incubation at 4 °C, anti-K-(NQTGG) antibody-bound beads were washed three times with 1 ml of 1 × PBS, twice with 1 ml of 0.1 × PBS, and once with double-distilled water. SUMO peptides were eluted three times with 200 μl of 0.2% formic acid in water and dried down by Speed Vac.

Cell pellets were collected and lysed in IP lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, and protease inhibitor) at 4 °C for 1.5 h. The equal cell lysate, primary antibody (1 μg), and 10 μl PureProteome protein A/G magnetic beads (Millipore) were incubated together at 4 °C overnight with gentle rocking. The beads were washed three times with IP wash buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Tween 20, pH 7.4). The bound protein complexes were then eluted by IP lysis buffer at 85 °C for 10 min. The complexes were fractionated on SDS-PAGE for immunoblotting analysis.

For in vitro ubiquitination, HEK293T cells were transfected with plasmids as described previously for 36 h. The cells were then treated with 15 μM MG132 (MedChemExpress, Monmouth Junction, NJ, USA) for 6 h and lysed in buffer on ice for 30 min with oscillation for 30 s at 5 min intervals. The samples were centrifuged for 10 min at 12,000 g at 4°C, and the supernatants were collected. A total of 0.5 mg of lysate was incubated overnight at 4°C with PureProteome™ Protein A/G Magnetic Beads (Millipore, Bedford, MA, USA) and antibodies against SIAH1 (Abcam, Cambridge, UK), RPS3 (Abcam, Cambridge, UK), GFP (Proteintech, Rosemont, IL, USA) or FLAG (Cell Signaling Technology, Danvers, MA, USA). After six washes with lysis buffer, the immuneprecipitates were resuspended in 5× loading buffer, degenerated and further analysed by Western blotting of cell lysates. Immunoblotting was performed using ubiquitin, SIAH1, RPS3, GFP, FLAG, and GAPDH antibodies.