M20002

For immunoblotting, we used the following affinity-purified antibodies: anti-Smad2/3 (1:1,000; 3102, Cell Signaling Technology), anti-p-Smad2 (1:500; AB3849, Millipore), anti-Smad3 (1:1,000; 9523, Cell Signaling Technology), anti-p-Smad3 (1:1,000; 04-1042, Millipore), anti-Fscn1 (1:5,000; 5535-1, Epitomics), anti-FoxA2 (1:1,000; 8186S, Cell Signaling Technology), anti-Nanog (1:1,000; Ab80892, Abcam), anti-Sox17 (1:1,000; 09038, Millipore), anti-Actin: (1:1,000; CW0096A, CWBIO), anti-Tubulin (1:1,000; CW0098A, CWBIO), anti-Myc (1:3,000; M20002, Abmart), anti-HA (1:3,000; M20003, Abmart) and anti-Flag (1:5,000; F3165, Sigma). Anti-Myc (1:100) and anti-HA (1:100) antibodies were also used in immunoprecipitation assays.
For each immunoprecipitation assay, either HEK293T or HEK293 cells were transfected with indicated plasmids. Cells were collected 48 h after transfection and lysed with TNE lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40) containing a protease inhibitor cocktail. Lysates were incubated with protein A sepharose beads and indicated antibodies at 4 °C for 4 h. The beads were then washed four times with TNE buffer. The bound proteins were separated by SDS–PAGE and visualized by western blotting. The intensity of each band was quantified using Image J. Full images of uncropped western blots are shown in Supplementary Fig. 10.

The CDS of ATG6 and MAPK20 was introduced into the pFGC1008-HA and pFGC5941-MYC vector, respectively, and transformed into A. tumefaciens strain GV3101. After inoculation into N. benthamiana leaves for 2 d, total proteins were extracted with isolation buffer [100 mM HEPES (pH 7.5, Solarbio, Beijing, China, H8090), 5 mM EDTA, 5 mM EGTA (Solarbio, E8050), 10% glycerol, 10 mM Na₃VO₄ (Solarbio, G8460), 10 mM NaF, 50 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA, G9422), 1 mM PMSF, 1 × protease inhibitor cocktail tablet (MedChemExpress, Monmouth Junction, NJ, USA, HY-K0011), 7.5% polyvinylpolypyrrolidone (Solarbio, P8070)] and were centrifuged at 12000 g, 4°C for 15 min. The proteins were immunoprecipitated with anti-HA magnetic beads (MedChemExpress, HY-K0201). ATG6-HA and MYC-MAPK20 were analysed with an anti-HA (Abmart, M20003) and anti-MYC antibody (Abmart, M20002), respectively.

To examine the binding region of GluAs with ABHD6 in HEK293T cells, 7.5 μg full-length GluAs or GluAs deletion plasmids, together with 2.5 μg ABHD6, were transfected in a 60-mm dish. A pCAG empty vector was used as control. The cells were harvested 48 h after transfection. The HEK293T cells were washed with PBS once, kept at -80°C overnight, and thawed at 37°C for 1 min. Then, the cells were collected with PBS and centrifuged at 17,000× g for 1 min at 4°C to obtain the cell pellets. 200 μl of buffer A (150 mM NaCl, 20 mM HEPES, 2 mM CaCl₂, 2 mM MgCl₂, 0.1 mM EDTA, 1% Triton, and protease inhibitors) was added to the cell pellets. Proteins were solubilized by gentle rocking at 4°C for 2 h. Next, the insoluble fractions were removed by centrifugation at 17,000× g for 30 min. A total of 150 μl of supernatant was used for an affinity chromatography assay, and 16 μl were used as inputs. 3 μl anti-myc antibodies (M20002, Abmart) and 24 μl of protein G beads were added to samples and rotated overnight at 4°C. Then, the beads were washed five times with wash buffer (150 mM NaCl, 20 mM HEPES, 2 mM CaCl₂, 2 mM MgCl₂, 0.1 mM EDTA, 1% Triton, and protease inhibitors), boiled in SDS sample buffer, and subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis.

To detect that CYC U2 can interact with OsGSK2, the rice were ground to a fine powder in liquid nitrogen and solubilized with 2× extraction buffer (100 mM Tris-HCl, pH7.5, 300 mM NaCl, 2 mM EDTA, pH 8.0, 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail). The extracts were centrifuged at 20,000 × g for 10 min, and the resultant supernatant was incubated with prewashed anti-FLAG M2 beads (Sigma-Aldrich) for 3 h at 4 °C, and then the beads were washed six times with the 2× extraction buffer. The immunoprecipitates were eluted with 1× SDS sample buffer, separated on a 12% SDS-PAGE gel, transferred to nitrocellulose membrane (Amersham Biosciences), and detected with corresponding antibodies. Anti-GSK2 (AbP80050-A-SE, 2000-fold dilution) was purchased from Beijing Protein Innovation.
To detect that CYC U2 can interact with D3, the full-length coding sequence of CYC U2 was cloned into pCambia 1306 vector which fuse with FLAG tag and D3 was cloned into the pCambia 1300 vector which fuse with MYC tag, and then transformed into the Agrobacterium strain GV3101 and coinjected into young leaves of N. benthamiana. The protein extraction referred to the above method. The anti-FLAG (M20008; Abmart, 3000-fold dilution) and anti-MYC antibody (M20002; Abmart, 3000-fold dilution) was used to test the results. Co-IP-related blots were shown in Supplementary Figs. 10, 11.

The purified Flag-tagged protein was mixed with the purified GST-tagged protein (or Myc-tagged APRF1) in 1 ml of pull-down buffer [20 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1 mM DTT and EDTA-free protease inhibitor cocktail (Roche)]. A 100-μl volume of the mixture was used as input, and the remaining mixture was incubated with 40 μl of Glutathione Sepharose^® 4B at 4°C with gentle rotation for 1 h. After the Glutathione Sepharose^® 4B was washed six times with 1 ml of pull-down buffer at 4°C, proteins bound on the beads were eluted with 200 μl of elution buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 20 mM glutathione and 1 mM DTT] at 4°C for 30 min. Finally, the eluted and input samples were boiled with SDS loading buffer and separated on SDS-PAGE gels for western blotting with Flag antibody (Sigma, F7425) and GST antibody (Abmart, M20007) or Myc antibody (Abmart, M20002).

After mild alkaline treatment, cells were boiled in a standard electrophoresis loading buffer similar as previously described (Kushnirov, 2000 (link)). The protein samples were split by SDS-PAGE and then transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell system. After incubation overnight at 4°C using a primary antibody, the blots were incubated with a secondary antibody (926-32211; LI-COR Biosciences, Lincoln, NE, United States). Then, an Odyssey infrared 740 imager (9120; LI-COR Biosciences) was used to scan the blots. H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K18me1, H3K23ac, H3K79me2, and H3K79me3 antibodies were obtained from EASYBIO (BE3281, BE3275, BE3224, BE3276, BE3291, BE3227, BE3301, and BE3302; Beijing, China). MYC and HA antibodies were purchased from Abmart (M20002 and M20003; Shanghai, China). FLAG antibodies were purchased from Bioregent Bio company (AB1027t; Beijing, China) and Abmart (M20008L; Shanghai, China). The Pgk1p polyclonal antibody was generated in rabbits.