Hrp conjugated anti gfp antibody

Western blot detection of GFP labeled proteins was performed as described [49 (link)]. Briefly, biomass harvested from microcolonies or giant colonies was disrupted using glass beads and proteins in cell lysates (4–25 µg in a well) were subjected to SDS-PAGE and proteins transferred to a PVDF membrane. GFP was detected by mouse monoclonal horseradish peroxidase (HRP)-conjugated anti-GFP antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The peroxidase signal was visualized using Super Signal West Pico (Pierce Biotechnology, Waltham, MA, USA) on Super RX medical X-ray film (Fomei, Hradec Králové, Czech Republic). Membranes stained with Coomassie blue were used as loading controls (Figures S1 and S2).

Immunoblotting was performed as previously described (Mazur et al., 2021 (link)). PVDF membranes were stained with 0.2% Ponceaus S for protein loading visualization. For the detection of GFP-conjugated protein, HRP-conjugated anti-GFP antibody (Santa Cruz Biotechnology, USA) diluted 1:1000 was used according to the manufacturer’s protocol.

JAZ7, JAZ7mEAR, JAZ5 and JAZ8 were constructed as described for Y2-H assays except JAZ7-R2, JAZ8-R2 or JAZ5-R2 (Supplementary Table S2) were used as reverse primers in order to remove stop codons and cloned into pDONRZeo plasmid (Invitrogen). The entry clones were recombined with the Gateway-compatible pEarleyGate 101 (with 35S promoter and C-terminal YFP fusion tag), and GFP cloned into pEarleyGate100 used as control (Earley et al., 2006 (link)). TPL was amplified from Col-0 gDNA and cloned into pICH47742 (with 35S-promoter and C-terminal 4×Myc fusion tag) using the Golden Gate assembly method (Engler et al., 2008 (link)). Five-week-old N. benthamiana plants were used for Agrobacterium tumefaciens-mediated transient expression of indicated constructs as described previously (Cevik and Kazan, 2013 (link)). Co-immunoprecipitation experiments were carried out as described previously (Sohn et al., 2012 ). Leaf samples were harvested 2 d post-inoculation, and total protein extracts were incubated with 20 μl GFP-affinity matrix (Chromotek) for immunoprecipitation. HRP-conjugated anti-GFP antibody (Santa Cruz) and anti-c-Myc antibody (Santa Cruz) were used for immunoblot analyses.