Gfp trap a beads

Protein pulldowns employing GFP-trap_A beads (Chromotek) were conducted as described earlier [98 (link),143 (link)] with some alterations. A. nidulans strains were inoculated in a concentration of 5*10⁸ spores in 500 ml MM. Mycelia were harvested and immediately frozen in liquid nitrogen. Frozen mycelia were ground with a table mill in liquid nitrogen. Ground mycelia were mixed with B⁺ buffer in a ratio of 1:1 and centrifuged twice for 20 min at 4000 rpm at 4°C. Supernatant was filtered through 20 μm sterile filters (Sartorius) and mixed with 1:100 GFP-trap_A beads (Chromotek) and incubated o/n at 4°C.

Ubiquitination status of GFP-tagged proteins was assessed by using ubiquitination assay^{24 (link)}. HEK293T cells, transfected with HA-ubiquitin, GFP-substrates, and USP32 or its mutant as indicated, were lysed in 300 µL lysis buffer 1 (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100, 10 mM N-methyl maleimide (general DUB inhibitor diluted in DMSO, freshly added) and protease inhibitors (Roche Diagnostics, EDTA-free, freshly added) by scraping. Then, 100 µL lysis buffer 2 (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 2% SDS) were added to the crude lysates; samples were sonicated (Fisher Scientific FB120 Sonic Dismembrator, 3 pulses, amplitude 40%) and SDS was subsequently diluted by bringing sample volume to 1 mL with lysis buffer 1. After centrifugation (20 min, 4 °C, 20,817× g), lysates were incubated with 6 µL GFP_Trap_A beads (Chromotek) overnight at 4 °C. Beads were washed three times with lysis buffer. During the fourth washing step, 30 µL Protein G4 fast flow (GE Healthcare) was added and all liquid was removed prior to the addition of SDS sample buffer (containing 10 mM DTT). Proteins were denatured by heating at 95 °C for 15 min, subjected to 8% SDS-PAGE, and detected by Western blotting, as indicated.

Coimmunoprecipitation procedures were performed as reported previously [8 (link)], using GFP_Trap_A beads (Chromotek, Munich, Germany). Various GFP and mCherry fusion proteins [8 (link),12 (link)] were used as negative controls in confocal microscopy and coIP assays. Some proteins were specifically selected based on their ability to generate cytosolic aggregates (MLP124111), their similarity in size and accumulation pattern to tested proteins (MLP124202ΔCT), or their ‘stickyness in coIP/MS assays’; i.e. ability to associate with a high number of proteins, (PST10977 and PST03196). Sample preparation, liquid chromatography / tandem mass spectrometry (LC-MS/MS) and data analyses were performed as described in [8 (link)], using a hybrid mass spectrometer LTQ-Orbitrap XL (ThermoFisher Scientific, Carlsbad, California, USA) and a nanoflow-UHPLC system (NanoAcquity Waters Corp., Burnsville, Minnesota, USA). LC-MS/MS data were processed and plant interactors were scored as previously described [8 (link)] (Sheet B in S1 Table). Briefly, scores were calculated following the formula "protein score = maximal peptide count/(redundancy)²”. The redundancy value was calculated by integrating the coIP/MS data from [8 (link)].

Total proteins were extracted from N. benthamiana leaves 3 days after agroinfiltration of GFP::AVRcap1b or GFP::PexRD54 [74 (link)] and subjected to immunoprecipitation using GFP_Trap_A beads (Chromotek, Munich, Germany), as described previously [50 (link),97 (link),100 (link)]. PexRD54 was included as a control, as it is also a large P. infestans RxLR effector and extensive studies suggests that its role is likely independent of the NRC network [74 (link),75 (link)]. Final OD₆₀₀ for each protein is indicated in S13 Table. Immunoprecipitated samples were separated by SDS-PAGE (4% to 20% gradient gel, Biorad, United Kingdom) and stained with Coomassie brilliant Blue G-250 (SimplyBlue Safe stain, Invitrogen). Enriched protein samples were cut out of the gel (approximately 5 × 10 mm) and digested with trypsin. Extracted peptides were analysed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with the Orbitrap Fusion mass spectrometer and nanoflow-HPLC system U3000 (Thermo Fisher Scientific, UK) [48 (link),50 (link)]. A total of 3 biological replicates for each sample type were submitted.

The Co-IP protocol was described previously [56 ]. IP was performed by affinity chromatography with GFP_Trap_A beads (Chromotek). Proteins were eluted from the beads by heating 10 minutes at 70 °C. Proteins were separated by SDS-PAGE and were transferred onto a polyvinylidene diflouride membrane using a Trans-Blot turbo transfer system (Bio-Rad, Munich). The membrane was blocked with 5% milk in Tris-buffered saline and Tween 20. GFP detection was performed in a single step by a GFP (B2):sc-9996 horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA); RFP detection was performed with a rat anti-RFP 5F8 antibody (Chromotek, Munich) and an HRP-conjugated anti-rat antibody. Membrane imaging was carried out with an ImageQuant LAS 4000 luminescent imager (GE Healthcare Life Sciences, Piscataway, NJ). Instant Blue (Expedeon, Cambridge) staining of rubisco was used as a loading control.

Differentiated SH-SY5Y cells were transfected with 8μg GFP-C1-Tau3R/4R plasmid or control GFP plasmid. 48 hrs after transfection, proteins were extracted with lysis buffer (Pierce, Rockford, IL) with added protease inhibitor (11255500, Roche, Mannheim, Germany). Immunoprecipitation (IP) was performed with GFP-Trap A beads according to the provided protocol (ChromoTek Inc., Planegg-Martinsried, Germany). 2.3μg of NAP, diluted into lysis buffer (NAP 2.3μg/sample), or the equal volume of lysis buffer w/o NAP were added to lysates of the transfected SH-SY5Y cells. Protein lysate with or without NAP was added to equilibrated GFP-Trap A beads and incubated 2 hrs at +4°C under constant mixing. Flow-through, wash 1 and 3, and elution fractions were collected and analyzed by immunoblotting with the appropriate antibodies.