α myc

Constructs were generated by PCR and cloned into pAc, pMT, pcDNA3 (Invitrogen) or pEF6 and verified by sequencing. Specific point mutations were generated using site-directed mutagenesis with Pfu Turbo polymerase (Stratagene). The Ub-sgg10 fusion construct was generated by PCR using Easy-A polymerase and cloned into pMT-V5/His. The following antibodies were used: α-MYC (Sigma, M5546, 1:2,000), α-HA (Roche, 11867423001, 1:2,000), α-V5 (Serotec, MCA1360, 1:2,000), α-RIPK1 (BD Biosciences, 610459,1:1,000 for western blotting (WB) and 1:50 for immunofluorescence studies), α-Actin (Santa Cruz, sc-1615, 1:4,000), α-MYO7A (Developmental Studies Hybridoma Bank, 138-1-s, 1:1,000 for WB and 1:50 for immunofluorescence studies), α-CASP8 for WB (MBL, M032-3, 1:5,000), α-CASP8 to detect cleaved CASP8 (for human: R&D, AF1650, 1:2,000; for mouse: Cell Signaling Technology, 9429, 1:1,000), rabbit and goat α-CASP8 (Santa Cruz Biotechnology, sc-7890 and sc-6136, both 1:50 for immunofluorescence studies), CF488A-donkey α-mouse IgG (Biotium, A21202, 1:1,000), CF633-donkey α-rabbit IgG (Biotium, 20125, 1:1,000) and CF633-donkey α-goat IgG (Biotium, 20127, 1:1,000). SM (SM164) was a gift from Shaomeng Wang (University of Michigan) and TNF was obtained from Enzo Life Sciences. Uncropped WBs are shown in Supplementary Fig. 5.

RP subcellular localization was analyzed by immunofluorescence: a total of 1.5x10⁶ cells were centrifuged at 1,400 x g for 5 minutes at RT, followed by washing with 500 μl of PBS. Cells were fixed for 10 minutes at RT in 500 μL of 3% paraformaldehyde in PBS, pelleted and washed once with 500 μL PBS. The pellet was resuspended in 100 μL of 0.1% glycine in PBS and 30 μL of the total cells were added to poly-lysine slides and left to adhere for 30 minutes. Then, fixed cells were permeabilized with 30 μl of 0.2% Triton X-100 in PBS solution for 5 minutes at RT, then washed 5x in 1x PBS. Blocking was performed for 30 min with 5% skimmed milk powder dissolved in TBS-T. α-myc (Sigma C3956) primary antibody was diluted in blocking solution (1:4000) ratio and incubated for two hours. After incubation, five washes were performed with 1x PBS then secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen A11001) were 1:500 diluted in PBS and incubated for 30 minutes at RT protected from the light. Nuclei and kinetoplast staining were performed using HOESCHT (Invitrogen H3570) at 2 μg·mL^-1 for 15 min. Images were acquired on a Leica DMI6000B fluorescence microscope at 60x magnification and processed using the Fiji Image software J (https://imagej.net/Fiji/Downloads) [24 (link)].

Proteins were separated on SDS-PAGE, transferred to the PVDF membrane (Millipore) and detected using one of the following antibodies: α-GFP (mouse, 1:1,000, Roche), α-RFP (rat, 1:1,000, Chromotek), α-Myc (1:1,000, Sigma), α-ARF1 (rabbit, 1:2,000, Agrisera), α-GN-SEC7 (rabbit, 1:2,500) [37 (link)], and POD-conjugated secondary antibodies. Chemiluminescence signal was acquired using Fusion Fx7 detection system (PEQlab, Erlangen, Germany).

For western blot analysis, samples of 10 OD of cells were harvested, washed once with TBS and resuspended in 120 μl lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 1% NP-40, 0.5% sodium-deoxycholate, 0.1% SDS, 10 mM sodium fluoride 0.5 mM EDTA, 10% glycerol, 1.5 μl sodium-butyrate (5 mM) and protease inhibitors). The proteins were extracted by vortexing 5 minutes at 4°C with acid-washed glass beads. 40 μl of 4xLämmli buffer was added to each sample, and samples were heated for 3 min to 95°C. Protein amounts equivalent to 1 OD cells were separated on SDS-PAGE gels (bottom: 15%, top 10%) and analysed by Western blotting. Antibodies used for western blotting were α-H4 K16Ac (Millipore 05–1232), α-myc (Sigma M4439) and α-GAPDH (loading control, Abcam ab9385). The immunoblots were imaged on a Bio-Rad imaging system.

Five hundred milligrams of leaf material were mixed with 2 ml of extraction buffer (50 mM HEPES pH 7.3, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA pH 8, 5 mM DTT, 1% PVPP, and 1× Protease Inhibitor Cocktail [Sigma, P599]) and centrifuged for 10 min at 14,000 × g at 4 °C; 5× Laemmli sample buffer was added to 100-µl supernatant and boiled for 5 min. Equal amounts of supernatant were loaded on 12% SDS–PAGE gels. Antibodies used for immunoblotting were as follows: α-GFP-HRP (1:5,000 Miltenyi Biotec), α-RFP-HRP (1:5,000 Abcam), α-myc (1:10,000, Sigma-Aldrich), α-actin (dilution 1:5,000, Agrisera), α-Hsp90-1 (1:2,000 Abcam), and α-polyQ (1:1,000 Merck).