H3663 200ul

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.

Western blots were carried out as previously described^{26 (link)}. Primary antibodies used were anti-HA (Sigma, H3663-200UL; 1:1000 dilution), anti-FLAG (Sigma, F1804-200UG, 1:2000 dilution) and secondary antibody Goat anti-Mouse IgG1, HRP from Thermo Fisher Scientific, PA1 74421, (1:10000 dilution). Full scan uncropped Western blots are provided (Source Data 1). Immunolocalization of RAP2.3 was carried as described^{70 (link)}. Four-day-old seedlings containing the 35S:RAP2.3^3XHA transgene were treated for 3 h with 50 μM bortezomib or DMSO (control). Anti-HA primary antibody (Roche, Lewes, UK) and Alexa-Fluor-488-coupled anti rat secondary antibody (1:200 dilution) (Molecular Probes, Carlsbad, CA) were used for detection. Seedlings were counter stained using Propidium Iodide and visualised using confocal microscopy.

Proteins were extracted from mycelia grown under the specified conditions by being ground to a fine powder under liquid nitrogen and resuspended in buffer B250 as described by Assis et al. (73 (link)). Samples were incubated with 20 μl of anti-HA antibody H3663-200UL (Sigma)-loaded magnetic beads or with 20 μl GFP-Trap (Chromotek) resin for 4 h on a horizontal shaker. Magnetic beads were washed two times with buffer B250 and once with protein extraction buffer (73 (link)) before samples were incubated with 20 μl SDS-sample buffer and incubated at 95˚C for 5 min. Samples were run on a 12% SDS-PAGE gel, and Western blotting was carried out as described previously (20 (link)).