Anti gfp hrp

Constructs were co-expressed in N. benthamiana leaves by Agrobacterium-mediated transient expression (as above). After 2–3 days, plant proteins were extracted from harvested leaves in 5 ml of extraction buffer as described earlier⁴⁸ . Proteins were incubated with agarose or sepharose beads of indicated epitope tags for 2 h at 4 °C. For GFP and Myc IPs, GFP-Trap and Myc-Trap agarose beads (Chromotek) were used and for FLAG and HA IPs, anti-FLAG M2 agarose and anti-HA agarose (Sigma) beads were used. After washing with washing buffer five times, beads were resuspended with 50 μL of 1 × SDS loading buffer. Proteins were separated on 10% SDS-PAGE gel, transferred onto a PVDF membrane using a semi-dry electroblotter (Bio-Rad), and detected with antibodies. Mouse anti-FLAG (1:5000, Sigma), anti-cMyc-HRP (1:10000, Santa Cruz), anti-HA-HRP (1:1500, Santa Cruz), anti-GFP-HRP (1:10000, Santa Cruz), rabbit anti-phospho-p44/42 MAPKs (1:5000, Cell Signaling Technology) and rabbit anti-14-3-3 (1:2000, Argisera) were used. The chemiluminescent signal was detected using Amersham ECL-Plus blotting detection system (GE Healthcare). For Blue native PAGE, proteins were extracted in the same way, separated on an Invitrogen 4-16% gradient NativePAGE Bis-Tris Gel, and transferred onto a PVDF membrane following the manufacturer’s instructions.

Immunoprecipitated or total extracts were separated on a 12% SDS-PAGE and transferred to a PVDF membrane (Millipore, Mississauga, Canada). Membranes were blocked in PBS/5% milk or PBS/0.3% BSA and incubated with anti-HBZ (1:2,000; Gaudray et al., 2002 (link)), anti-B23 (1:500, Sigma-Aldrich Canada Co., Oakville, Canada), anti-C23 (1:500, #sc-13,057, Santa Cruz Biotechnology Inc.), anti-DsRed (1:1000,#sc-101,526, Santa Cruz Biotechnology Inc), anti-GFP-HRP (1:1,000, #sc-9,996, Santa Cruz Biotechnology Inc.), anti-Tubulin (1:5,000, #T5168, Sigma-Aldrich Canada Co.), anti-β-Actin (1:5,000, #sc-47,778, Santa Cruz Biotechnology Inc.), anti-Tax (1:2,000, #sc-57,872, Santa Cruz Biotechnology Inc), or anti-Myc (1:250) antibodies overnight. After several washes, membranes were incubated with HRP-conjugated sheep anti-rabbit IgG antibodies (1:5,000, #5220–0336, Seracare, Milford MA) or anti-mouse IgG antibodies (1:5,000, #5220–0338, Seracare) for 2 h, washed several times and incubated with the BM Chemiluminescence Blotting Substrate. Membranes were analysed using the Fusion FX7 device.

Small leaf segments of N. benthamiana or A. thaliana seedlings expressing SUBEX-GFP or SUBEX-C57Y-GFP were incubated in liquid 0.5× MS medium supplemented with 1% sucrose and with or without 50 μM kifunensine, 100 μM clasto-lactacystin β-lactone (Sigma-Aldrich) or 50 μM MG132 (Sigma-Aldrich) for 5 h in the dark. Subsequently, proteins were extracted with 1× PBS containing 1% Triton X-100 and 1% protease inhibitor cocktail and subjected to SDS/PAGE and immunoblotting with anti-GFP-HRP, anti-ubiquitin (P4D1, Santa Cruz Biotechnology) and anti-protein disulfide isomerase (PDI) [27 (link)] antibodies. Detection was done with a ChemiDoc imager (Bio-Rad Laboratories) and quantification with Quantity One software (Bio-Rad Laboratories).