Anti myc

For protein expression analysis, total proteins were extracted from N. benthamiana tissues with buffer containing 150 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, 2 mM CaCl₂, 5 mM MgCl₂, 0.15% NP-40, 0.1% Triton X-100 (pH 7.5), 10 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride (PMSF), plant protease inhibitor cocktail (VWR), and 20 μM MG132. Protein samples were separated on 10% Bis-Tris PAGE gels, transferred electrophoretically to 0.45-μm Amersham Protran Premium nitrocellulose membranes (GE Healthcare), and detected with anti-GFP (Takara), anti-HA, anti-FLAG, or anti-Myc (BioLegend) antibodies.
For co-IP assays, protein extracts were incubated with an anti-Myc antibody (1:2000, Sigma-Aldrich) at 4°C for 16 h followed by incubation at 4°C for 4 h with protein A/G magnetic beads (VWR) equilibrated with the extraction buffer. The beads were washed six times with 1 ml of extraction buffer, and immunoprecipitated samples were analyzed by western blotting with anti-HA, anti-FLAG, or anti-Myc (BioLegend) antibodies.

Log-phase cells were harvested, washed, and stored at −80°C. Cells were disrupted by bead beating using a BeadBlaster 24 (Benchmark Scientific) in lysis buffer (0.2 M Tris base, 0.39 M ammonium sulfate, 10 mM MgSO₄, 1.0 mM EDTA, 20% glycerol, 1× Protease inhibitor cocktail at pH 7.9). The clarified lysate was dialyzed in dialysis buffer (20 mM HEPES, 10 mM MgOAc, 200 mM KOAc, 2 mM EGTA, 20% glycerol, 1× Protease inhibitor cocktail at pH 7.9) for 3–4 h at 4°C. Protein (0.75 mg) was diluted to 2 mg/mL using dialysis buffer and incubated with Not4 antiserum and protein-A sepharose overnight at 4°C. Beads were washed three times with immunoprecipitation wash buffer (dialysis buffer containing 0.03% NP-40) and once with NaCl immunoprecipitation wash buffer (25 mM Tris-HCL at pH 7.5 , 0.10 M NaCl, 2 mM EDTA, 10% glycerol, 0.03% NP-40). Samples were loaded onto 7.5% SDS-PAGE gel and transferred to membranes. Blots were probed with antimyc (BioLegend, 9E10) and Not4 antiserum. One-fifteenth of the input sample was loaded to the gel.