Pierce anti ha magnetic beads

IP was performed as previously described26 (link). Briefly, after transient transfection with plasmid, HEK-293T cells were lysed in ice-cold IP buffer (20 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease inhibitor cocktail tablets (04693132001, Roche). The cell lysates were incubated with the indicated antibody-conjugated beads (Anti-Flag M2 Magnetic Beads, M8823, Sigma; Pierce Anti-HA Magnetic Beads, 88836, Thermo Fisher Scientific) at 4°C overnight. The immunoprecipitates were subjected to immunoblotting using the indicated primary and corresponding secondary antibodies.

For affinity purification of RAP2.3^3XHA, seven-day old seedlings grown in continuous light were treated for 2 h with 50 μM bortezomib (dissolved in DMSO). Total protein was extracted by grinding 0.5 g tissue in liquid nitrogen with 700 μl of IP buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.05% IGEPAL, 50 μM BZ, 100 mM PMSF). Extract was centrifuged at 13,000 rpm for 30 mins and supernatant (600 μl + 200 μl TBST) was incubated with 25 μl of Pierce™ Anti-HA Magnetic Beads (Thermo Fisher, UK) for 30 mins at room temperature. After incubation flow through was collected and beads were washed three times for 2 mins each with 300 μl of PBST (Tris Buffered Saline Pack (25 mM Tris, 0.15 M NaCl; pH 7.2, Thermo Fisher, UK; 0.1% (v/v) Tween-20). Finally, beads were washed with 300 μl molecular grade water. Elution of the beads was achieved by adding 100 μl of SDS-PAGE Lane Marker Non-Reducing Sample Buffer (2X), (0.3 M Tris-HCl, pH 6.8, 5% (v/v) SDS) (Thermo Fisher, UK) to the tube containing beads, followed by incubation at 95 °C for 10 min. Two μl of β-mercaptoethanol was added before loading onto an SDS-PAGE gel. Western blot analysis of RAP2.3^3XHA abundance were carried out as previously described^{8 (link)}. Anti-ubiquitin (Agrisera AS08 307 1:4000 dilution) and anti-HA antibodies (Sigma, H3663-200UL; 1:10,000 dilution) were used for Western immunodetection.

After the transfection with p62-HA, the cells were starved for 48 h. After two washes with cold PBS, cells were lysed with lP lysis buffer (40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS, protease inhibitor cocktail P8340 Sigma and 1 mM PMSF). Protein extracts were incubated overnight at 4 °C with anti-HA magnetic beads (Pierce Anti-HA Magnetic Beads, Thermo Fisher #88836). Thereafter beads were washed twice with cold PBS and eluted with Laemmli buffer for immunoblot analysis.