α gfp

In Co-IP assays with proteins expressed in N. benthamiana Roq1 reconstitution assays, five 10 mm leaf discs were collected to form a sample. Total protein from the plant material ground to fine powder was extracted in 2 ml of the extraction buffer (10% glycerol, 100 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 300 mM NaCl, 10 mM DTT, 0.5% NP-40, 2% PVPP, 1x Plant protease cocktail (11873580001, MilliporeSigma)). Lysates were centrifuged for 35 min at 4500 × g and filtered through two layers of Miracloth (475855, MilliporeSigma). The 50 µl aliquots of the filtered supernatant were taken as input samples. Co-IP were conducted for 2 h with 12 μl α-HA affinity matrix (11815016001, MilliporeSigma) under constant rotation. Beads were collected by centrifugation at 4000 × g for 1 min and washed four times in extraction buffer (without DTT and PVPP). All co-IP steps were conducted at 4 °C. Beads and input samples were boiled at 95 °C in 100 μl 2 × Laemmli buffer for 10 min. Antibodies used for immunoblotting were α-GFP (11814460001, MilliporeSigma), α-HA (1:5000; c29f4, Cell Signalling), α-FLAG (1:5000; f1804, MilliporeSigma), HRP-conjugated antibodies (A9044 and A6154, Sigma-Aldrich; sc-2006 and sc-2005, Santa Cruz). Antibodies were used in dilution 1:5000 (TBST with 3% non-fat milk powder). Images of blots are provided in the Source data file accompanying this paper.

Nuclear extracts (600 μl) from the pEDS1:EDS1-YFP complementation line were processed as for IP input in the subsection “Immunoprecipitation (IP) of EDS1-YFP and TRB1-GFP from respective Arabidopsis complementation lines”. Obtained samples were fractionated on the column Superose 6 10/300 GL (50 kDa–5 MDa range, GE Healthcare Life Sciences, Ӓkta FPLC) at the rate 0.5 ml/min in 20 mM Tris–HCl pH 7.4 and 150 mM NaCl. The temperature was kept at 4 °C. In total, 28 0.5 ml fractions per sample were collected, concentrated with StrataClean resin (400714, Agilent) and analysed using Western blot method (α-GFP, 11814460001, MilliporeSigma) with the same total EDS1-YFP sample on each blot for the between-blot normalisation. A high-molecular weight marker was run prior to each experiment (28403842, GE Healthcare Life Sciences).

Proteins were separated by SDS-PAGE (10% or 4–20% Mini-PROTEAN TGX gels, BioRad, Hercules, CA, USA) followed by immunoblot analysis using mouse monoclonal α-HA (BioLegend, San Diego, CA, USA) or α-GFP (Millipore Sigma) at a dilution of 1:10,000 and polyclonal rabbit α-Rad53 (Abcam) antibodies at a dilution of 1:2000 to detect Pkc1-HA or -GFP, Hrr25-HA or -GFP and Rad53, respectively. Secondary goat anti-mouse (Jackson ImmunoResearch, Westgrove, PA, USA) and donkey anti-rabbit (GE Healthcare, Chicago, IL, USA) antibodies were used at a dilution of 1:10,000. SDS-PAGE gels used to detect Pkc1 band-shifts were 10% and those used for Pkc1 association with Hrr25 were 4–20%.

The following plasmids were used: human DVL2-GFP and FLAG-DVL2 (12 (link)); FLAG-DVL2, E499G, K446M (46 (link)), G436P, S418A, and S435A (11 (link)); and SNAP-FZD5 (47 (link)). Lip-DEP_402–510 was megaprimer cloned into Lip-DEP_416–511 (11 (link)). His₆ N terminally tagged LipDEP_416–511 (LipDEP_416–511-His₆) was cloned into a pNHD vector (36 (link)) with Gibson assembly, and an amber mutation (TAG) at position encoding for S435 was inserted by QuickChange cloning. DVL2 and DEP point mutations were generated by standard procedures and verified by sequencing. The following antibodies and resins were used: α-FLAG (Sigma) α-GFP (Sigma), α-actin (Abcam), α-SNAP (NEB), α-DVL2 (CST), and α-GSK3β (CST).

The following plasmids were used: human DVL2-GFP and FLAG-Axin (Fiedler et al., 2011 (link)); FLAG-DVL2, E499G, K446M, and D460K (Mund et al., 2015 (link)); and SNAP-FZD5 (Koo et al., 2012 (link)). DEP-RFP (red fluorescent protein) was generated by subcloning DEP into DsRed. DEP and FZD5 mutants were generated by standard procedures and verified by sequencing. The following antibodies and resins were used: α-FLAG, α-tubulin, and α-GFP (Sigma); α-actin (Abcam); and α-SNAP (NE Biolabs).