Gtma 20

The harvested HEK293T cells and the resulting lysate were kept on ice during the preparation at all time. Per eight million cells, 1 mL of RIPA buffer (10 mM Tris (pH = 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.1% (w/v) SDS, 1 % (v/v) Triton X-100, 1% (w/v) deoxycholate), supplemented with 2.5 mM MgCl₂, 100 U/mL benzonase (Merck Millipore 70746-3) and 1 × protease inhibitor cocktail (PIC, Roche 05056489001) on the day of preparation was used for the lysis. Cells were resuspended in RIPA buffer and lysed for 1 h at 4 °C on a tube rotator. Afterward, the lysate was centrifuged (10,000 × g, 15 min, 4 °C) and the supernatant containing the proteins was transferred into a new tube. To enrich GFP-tagged proteins, the lysate was immediately incubated with GFP Nano-Traps either on agarose beads (Chromotek gta-20, for in vitro activity assay) or on magnetic agarose beads (Chromotek gtma-20).

SGBS cells were electroporated using the Neon Transfection System (Invitrogen) with C-terminally tagged C19orf12 or GFP control. The plasmid was cloned from Origene rc231802 into the pegfp-N1 Gateway destination vector (gifted from R. Shaw, supplied by Addgene, plasmid #31796). Two days after transfection, cells were washed with ice-cold PBS and scraped in ice-cold Co-IP buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA and protein inhibitor cocktail (Roche)) supplemented with 0.5% Nonident P40. Lysates were incubated on ice for 30 min and cleared by centrifugation (4 °C, 10 min, 17,000g). The supernatant was diluted using 3× the volume of Co-IP buffer without detergent and incubated with washed anti-GFP magnetic agarose, 1:20 dilution (ChromoTek, gtma-20, lot 90122001MA)) for 1 h at 4 °C. Beads were washed with Co-IP buffer + 0.05% NP40 and Co-IP buffer without detergent. For the digest, beads were first incubated with 50 µl elution buffer I (2 M urea, 50 mM Tris/HCl, pH 7.5, 20 µg µl⁻¹ Trypsin (Sigma, t6567) and 1 mM dithiothreitol) for 30 min at 37 °C, 1,300 rpm. Afterwards, beads were incubated in 50 µl elution buffer II (2 M urea, 50 mM Tris/HCl, pH 7.5 and 5 mM chloroacetamide) in the dark. Supernatants were digested, combined overnight at 25 °C, 1,000 rpm and peptides were acidified using 1 µl trifluoroacetic acid and purified on C18 Stage Tips.

Cells were harvested with IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 20 mM N-ethylmaleimide (NEM), and 1% protease inhibitor cocktail), followed by sonication and pre-clearance using 1 mg/mL BSA-treated Sepharose. Supernatant collected from centrifugation were incubated with antibody-conjugated agarose beads for at least 4 h at 4 °C with rotation. The samples were centrifugated at 2500 × g for 2 min and washed the beads with IP buffer containing 0.1% NP-40. The following are the beads used for immunoprecipitation or pulldown analysis. Anti-Flag M2 affinity gel (A2220, Sigma-Aldrich), EZview Red Anti-HA affinity gel (E6779, Sigma-Aldrich), Glutathione Sepharose 4B (17-0756-01, GE healthcare), Ni-NTA Agarose (1018244, QIAGEN), and Protein G Sepharose 4 Fast Flow (GE17-0618-01, GE healthcare). Finally, the samples were suspended with 2× of Laemmli sample buffer and boiled at 95 °C for 3 min. All samples were analyzed by using Western blotting. For GFP trapping, cells were harvested with IP buffer containing 0.2% SDS. The supernatant was incubated with GFP-Trap Magnetic Agarose (gtma-20, ChromoTek) or Myc-Trap Magnetic Agarose (ytma-10, ChromoTek) for 1 h at 4 °C with rotation. Unprocessed images of Western blots are provided in the source data.

NTL8 protein ChIP experiments were carried out following the methods described in Zhao et al. (2020) (link). Briefly, nuclei were extracted from 3 g of materials with 30 mL of Honda buffer (0.4 M sucrose, 2.5% Ficoll, 5% dextran T40, 25 mM Tris-HCl at pH 7.4, 10 mM MgCl₂, 0.5% Triton X-100, 0.5 mM PMSF, proteinase inhibitor cocktail [Roche 04693159001], 5 mM DTT). After nuclei extraction, purified nuclei were lysed by RIPA buffer (1× PBS, 1% Igepal CA-630 [Sigma I8896], 0.5% sodium deoxycholate, 0.1% SDS, proteinase inhibitor cocktail) and then fragmented by sonication (Diagenode Bioruptor). After sonication, the fragmented chromatin extract was cleared by centrifugation at 13,000 rpm for 15 min at 4°C before immunoprecipitation. The immunoprecipitations were performed with GFP-trap beads (Chromotek GTMA-20) for GFP-NTL8-D2 (Fig. 2G) and anti-HA magnetic beads (Pierce 88836) for HA-NTL8 (Supplemental Fig. S4H) in the ntl8-OE3 line. Relevant primers are listed in Supplemental Table S3.

Total proteins were extracted using a lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA at pH 8.0, 0.1% Triton X-100, 0.2% NP-40, 0.6 mM phenylmethylsulfonyl fluoride (PMSF)) supplemented with a freshly added protease inhibitor cocktail (Roche LifeScience) and ΜG132 (10 μM). For the immunoblotting assay, the protein samples were separated on 10% SDS–PAGE and detected by antibodies including anti-GFP (1:2,000 dilution, ab32146, Abcam), anti-BRI1 (1:1,000 dilution, Setaria italica BRI1)^{34 (link)}, anti-TaBKI1 (1:1,000 dilution, prepared in this study, ABclonal), anti-SLR1 (1:1,000 dilution, A16279, ABclonal) and anti-GRF4 (1:1,000 dilution, A20348, ABclonal). For the co-IP assay, about 20 μl anti-GFP magnetic agarose beads (gtma-20, Chromotek) was incubated with protein samples for 3 h at 4 °C. The beads were cleaned four times with a wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8.0, 0.6 mM PMSF and 1× protease inhibitor cocktail), and the immunoprecipitated proteins were separated by SDS–PAGE and detected with anti-GFP and anti-MYC (1:2,000 dilution, CB100002M, California Bioscience) antibodies.

The in vitro ubiquitylation assay was carried out as described previously^{36 (link)}. In brief, the MBP–ZnF-CT and His–-BKI1 recombinant proteins were expressed and purified from Escherichia coli strain BL21 (DE3). MBP–ZnF-CT alone, or together with His–BKI1, was incubated with E1 (UBA1–GST, 50 ng), E2 (UBC10–GST, 50 ng) and Flag–ubiquitin (1 µg) in a reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM ATP, 20 mM MgCl₂ and 1 mM dithiothreitol) at 30 °C for 1.5 h. Similarly, for the assay of ZnF-mediated TaBKI1 ubiquitylation, TaBKI1 at different concentrations was incubated with MBP–ZnF-CT, E1 (UBA1–GST, 50 ng), E2 (UBC10–GST, 50 ng) and Flag–ubiquitin (1 µg) in reaction buffer. The reaction was stopped by adding SDS loading buffer. The obtained samples were boiled at 100 °C for 7 min, and the proteins were detected with anti-Flag (1:2,000 dilution, F1804, Sigma) antibody using SDS–PAGE. For the in vivo ubiquitylation assay, TaBKI1–GFP, ZnF–MYC and Flag–ubiquitin were co-expressed in N. benthamiana leaf epidermal cells. TaBKI1–GFP proteins were immunoprecipitated and purified to ubiquitin-conjugated TaBKI1–GFP through immunoblotting with anti-Flag (1:2,000 dilution, F1804, Sigma) antibody using anti-GFP magnetic agarose beads (gtma-20, Chromotek).

Wandering third instars (20 larvae) of each genotype (UH1>FMRP-YFP or Tubulin-GFP) were homogenized in 200 μL of RNase-free lysis buffer (20 mM HEPES, 100 mM NaCl, 2.5 mM EDTA, 0.05% (v/v) Triton X-100, 5% (v/v) glycerol) with 1% β-mercaptoethanol 1× protease inhibitor cocktail (complete mini EDTA-free Tablets, Sigma, 11836170001) and 400U RNase inhibitor (Applied Biosystems, N8080119). The supernatant was collected and diluted to 300 μL to reduce nonspecific binding. Next, the samples were incubated with GFP-trap coupled magnetic agarose beads (Chromotek, GTMA20) for 3 hours at 4°C. The bound beads were washed with lysis buffer (3X, 10 minutes). The bound RNA was purified by incubating the bead-protein-RNA conjugates with a 500-μL TRIzol and chloroform mixture (Ambion, 15596026) for 10 minutes at RT, followed by centrifugation. To precipitate RNA, glycogen (1 μL) and 2-propenol (250 μL) were added to the isolated aqueous layer. Finally, the precipitated RNA was reverse transcribed into single-strand cDNA using the SuperScript VILO cDNA synthesis kit (Thermo Fisher, 11754050) and then subjected to primer-specific PCR, with 2% agarose gels used to analyze the PCR products. All primers used in this study are summarized above in Table 2.