Briefly, harvested protoplasts were resuspended in 0.5 ml IP lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100 supplemented with 1 tablet of EDTA-free protease inhibitor cocktail (Roche) per 10 ml buffer). The cells were lysed by vigorous vortexing for 40 s followed by centrifugation in a microfuge at maximum speed for 10 min at 4 °C. The supernatant was incubated with 10 μl magnetic anti-HA beads (Thermo Fisher Scientific) for 3 h at 4 °C followed by three washes with IP lysis buffer. The beads were finally resuspended in 50 μl 2X SDS sample buffer.
Immunoblot assays were performed following standard procedures. Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen), transferred onto PVDF membrane (Fisher Scientific), and blocked in TBS-T buffer with 5% non-fat dry milk at room temperature for 1–2 h. Blots were incubated with primary antibody in TBS-T buffer with 5% non-fat dry milk at 4 °C overnight. The antibodies included in this study are α-SGT1b (this study), α-COI1 and α-HSP70 (AgriSera), α-HSP90 (Santa Cruz), α-FLAG (Sigma-Aldrich), α-HA (Roche) and α-HistoneH3 (Abcam).
Immunoblot assays were performed following standard procedures. Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen), transferred onto PVDF membrane (Fisher Scientific), and blocked in TBS-T buffer with 5% non-fat dry milk at room temperature for 1–2 h. Blots were incubated with primary antibody in TBS-T buffer with 5% non-fat dry milk at 4 °C overnight. The antibodies included in this study are α-SGT1b (this study), α-COI1 and α-HSP70 (AgriSera), α-HSP90 (Santa Cruz), α-FLAG (Sigma-Aldrich), α-HA (Roche) and α-HistoneH3 (Abcam).