Anti ha 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.

Leaf samples were processed as described earlier [28] (link). Samples were centrifuged at 13000 g for 20 min at 4°C, adjusted to 2 mg/ml total protein concentration, and pretreated with Protein A-agarose (Roach) for 3 to 4 h. Immunoprecipitations were performed on 1.5 ml total protein by adding anti-HA (Santa Cruz; 1∶100) or anti-GFP (Roach; 1∶100) overnight at 4°C. After incubation with 20 µl protein A-agarose at 4°C for 3 to 4 h, beads were washed 4 times with Tris-buffered saline (TBS) containing 0.5% (v/v) ND-40, immunoprecipitates were analyzed by immunobloting.
Samples were electrophoresed on 8% SDS-acrylamide gels, transferred onto nitrocellulose membranes (BIO-RAD), blocked, incubated overnight with primary antibody [anti-GFP (Roach) 1∶5000; anti-HA-HRP (Santa Cruz) 1∶2000], and washed in TBST (TBS with 0.1% (w/v) Tween-20). For anti-GFP, blots were incubated with a secondary antibody anti-mouse-HRP [(Santa Cruz) 1∶5000]. Signals were visualized using chemiluminescent substrate (Thermo Scientific) before exposure to X-ray film.