A2220

HDAC4-FLAG^{61 (link)} (gift from Eric Verdin; Addgene plasmid #13821) was transfected into HEK293 cells with PEI, and the cells were collected 2 days later, washed with PBS, and lysed in a hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT (Sigma), with PIC) at 4 °C with agitation for 1 hour. Extracts were pelleted by centrifuging at 17,000 g/10 min at 4 °C, and the supernatant was retained for use in the pulldown. M2-FLAG-agarose slurry (25 µl agarose slurry per IP; Sigma, A2220) was washed once with PBS before the beads were blocked with 1% BSA for 2 hours at 4 °C with agitation. The beads were then washed twice with PBS, before incubating at 4 °C with agitation for 1 hour with two milligrams of the HEK lysate containing overexpressed HDAC4-FLAG, before AAV9 (1 × 10¹² vg) or AAV9-138-Biotin (1 × 10¹² vg) was then added to each pulldown, and gently agitated overnight at 4 °C. Beads were washed 6 times with a wash buffer (PBS + 1% NP-40), before being resuspended in 2X Laemmli buffer with 5% β-mercaptoethanol and boiled for 5 minutes to elute the bound proteins.

Isolated crude mitochondrial samples (3.5–5 mg) were resuspended in digitonin solubilization buffer (1% [v/v] digitonin, 50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 1x PI [Roche]) at 2 mg/ml and incubated, end-over-end at 4 °C for 30 min. Solubilized mitochondria were clarified by centrifugation at 16,000g for 10 min at 4 °C. The supernatant was diluted to a final detergent concentration of 0.1% digitonin and incubated with pre-equilibrated anti-FLAG resin (Sigma Aldrich- A2220; 5–10 μl of resin/mg mitochondrial protein), end-over-end for 30 min at 4 °C. Resin and bound proteins were transferred to a Pierce spin column (Thermo Fisher Scientific-69705) and washed 8× in solubilization buffer containing 0.1% [w/v] digitonin. Bound proteins were eluted in 0.2 M glycine (pH 2.5) over 2 by 5 min incubations and elution fractions pooled. Total, supernatant, unbound, wash, and elution fractions were TCA precipitated, and pellet fraction directly resuspended into SDS-PAGE loading dye. All fractions were analyzed by SDS-PAGE and immunoblotting or elution fraction prepared for analysis by mass spectrometry.

Full-length human FAM46A/FAM46C with a FLAG-tag and BCCIPα/BCCIPβ with a Myc-tag were cloned into the pRK5 vector (556104, BD PharMingen). The plasmids were transfected into HEK293T cells with Lipofectamine 3000 (L3000001, Invitrogen) according to the manufacturer’s instruction. Cells were harvested 36 hours after transfection and lysed in the lysis buffer containing 50 mM tris (pH 8.0), 150 mM NaCl, 0.1% NP-40, 1 mM DTT, and protease inhibitor (78442, Sigma-Aldrich). Lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4°C. anti-FLAG beads (A2220, Sigma-Aldrich) were added to the supernatant, and the mixture was rotated at 4°C for 1 hour. The beads were washed with a lysis buffer three times. Proteins remaining on the beads were resolved with 4 to 20% gradient SDS-PAGE (XP04205BOX, Invitrogen) and detected by Western blot using anti-FLAG (F7425, Sigma-Aldrich) and anti-Myc antibodies (2278, Cell Signaling Technology).

Cells were transfected with Flag-GSK3β, Flag-hnRNPK, siRNA target against GSK3β or hnRNPK, or combined with treatments of TRAIL (Sino Biological Inc. Beijing, China), LiCl (Sigma), Gö6983 (1 μM, Beyotime, China), Rottlerin (6 μM, Millipore, USA), or CHX (10 μg/ml, Amresco, USA) when it is indicated. Cells were then harvested and lysed with lysis buffer (20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, leupeptin, and 1% protease inhibitor cocktail (Roche)). Whole cell lysates were separated by 10% or 12% SDS/PAGE.
Blots were probed with the specific antibodies against Flag (A2220, Sigma), GSK3β (27C10, Cell Signaling Technology), phospho-GSK3β (Ser9) (Cell Signaling Technology), hnRNPK (sc-28380, SANTA CRUZ), GAPDH (ZS-25778, ZSGB-BIO, China), β-catenin (Beyotime, China), Cleaved Caspase 3 (9664, Cell Signaling Technology), Cleaved Caspase 8 (9496, Cell Signaling Technology), c-FLIP (7F10, Alexis, Enzo life science, Switzerland). Horseradish peroxidase-conjugated secondary antibodies (ProteinTech Group) and enhanced chemiluminescence (ECL kit, Beyotime, China) were used to detect the expression of proteins.

Cytosol/membrane fractionation assay was performed according to a previously described method (Baghirova et al., 2015 (link)). Briefly, HEK 293T cells were harvested by trypsin treatment and washed with ice-cold PBS. Fractionation buffer FA (150 mM NaCl, 50 mM HEPES pH 7.4, 100 μg/ml digitonin (D141, Sigma), and 1 M hexylene glycol) supplemented with 1x protease inhibitor cocktail (P8340, Sigma, 100x stock solution) was added to the harvested cells to release cytosolic proteins. After incubation for 15 min on an end-to-end rotator at 4 ˚C, samples were centrifuged at 2,000 g for 10 min. The resulting supernatant was transferred and marked as cytosolic proteins. Fractionation buffer FB (150 mM NaCl, 50 mM HEPES pH 7.4, 1% (v/v) Nonidet-P40, and 1 M hexylene glycol) supplemented with 1x protease inhibitor cocktail (P8430, Sigma, 100x stock solution) was added to the resulting cell pellets to release membrane proteins. After incubation for 30 min on ice, samples were centrifuged at 7,000 g for 10 min. The resulting supernatant was transferred and marked as membrane proteins. Both cytosolic and membrane fractions were further immunoprecipitated using a FLAG-M2-affinity gel (A2220, Sigma, RRID:AB_10063035) and analyzed by immunoblotting. These experiments were repeated twice.

Two grams of transgenic roots expressing 35 S:GmRR11d-FLAG, pEarlygate100-FLAG, 35 S:GmNSP1a-GFP, 35 S:GmRR11d-GmNSP1a-GFP and 35 S:GFP were used for ChIP assays. Roots were cross-linked with 1% formaldehyde at 4 °C for 10 min and neutralized with 0.125 M glycine. Roots were ground to fine power in liquid nitrogen and the nuclei isolated. Immunoprecipitation was done using anti-GFP (ab290, Abcam) or anti-FLAG (A2220, ANTI-FLAG M2 Affinity Gel, Sigma) antibodies. Chromatin precipitated without the use of antibodies was used as a negative control, while chromatin isolated before precipitation was used as an input control. qPCR analysis was performed, and soybean ELF1b (GmELF1b) was used as an internal control. The specific primers used in this experiment are listed in Supplementary Table 1. Three independent biological repeats were performed for each analysis^{41 (link)}.
For the effect of exogenous cytokinin treatment on the binding activity of GmRR11d on the GmNIN1a promoter, two grams of transgenic roots (10 DAI) expressing EV1 and 35 S:GmRR11d-FLAG were treated with 0.1 μM 6-benzylaminopurine (BAP) for 4 h.