Agarose a g beads

HLMECs were lysed using a RIPA buffer (Thermo Fisher Scientific Inc.) with a protease inhibitor cocktail and a phosphatase inhibitor cocktail. The cell lysates were incubated with the anti-VEGF-A antibody (2 μg), rhVEGF-A (1 μg), and/or 5 μM 6,8-DG at 4 °C overnight. A total of 50 μL of the agarose A/G beads (Santa Cruz Biotech. Inc.) were added and rotated at 4 °C for 3 hr. After washing the beads with cold PBS buffer, proteins were removed from the beads in 30 μL 2× sample buffer and analyzed by 6% SDS-PAGE and Western blotting using anti-VEGFR-1 and anti-VEGFR-2 antibodies (Santa Cruz Biotech. Inc.).

Histone deacetylase (HDAC) activity assays were performed using a colorimetric system from Biovision (San Francisco, CA, USA) in accordance with the manufacturer’s protocol. To perform assays for specific HDACs—the HDAC1, HDAC2, HDAC3, and HDAC8—each protein was immunoprecipitated in the cell lysates using the antibodies against the respective proteins. Immunoprecipitated complexes were collected with agarose A/G beads (Santa Cruz, Dallas, TX, USA) and after washing with HDAC assay buffer (10% glycerol, 50 mM Tris (pH 8.0), and 0.1 mM ethylenediaminetetraacetic acid (EDTA)) used for assays.

The pJMJ29:JMJ29-GFP/jmj29-1, pELF3:ELF3-MYC/elf3-1, jmj29-1, and elf3-1 plants [22 (link),23 (link)] were used for ChIP assays. Plant materials used for the experiments were grown under NDs for 2 weeks. Harvested plant materials were fixed in 1% formaldehyde for 20 min with vacuum infiltration and ground in liquid nitrogen. Chromatin solubilized by the nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS, 1 mM PMSF, and 1× PIs) was sonicated at 4 °C to generate ~500 bp fragments using a Bioruptor Pico (Diagenode). The chromatin solutions were ultrasonicated for 10 cycles (30 s ON and 30 s OFF for each cycle on full power). The antibodies, including anti-GFP (ab290, Abcam, Cambridge, UK), anti-MYC (05-724, Millipore, Billerica, USA), anti-H3 (04-928, Millipore, Billerica, MA, USA), anti-H3K4me3 (07-473, Millipore, Billerica, USA), and anti-H3K9me2 (ab1220, Abcam, Cambridge. UK), and agarose A/G beads (SC-2003, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for ChIP. The fragmented DNAs were purified using a DNA elution kit. Precipitated DNA level was quantified by quantitative real-time PCR using specific primer sets listed in Supplementary Table S2. ChIP-qPCR values were normalized as a percentage of the input DNA.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotic solutions were purchased from HiMedia, Inc. (Mumbai, India). Lipofectamine RNAiMAX, Lipofectamine 3000, and pre-stained protein ladder, 10 to 250 kDa were purchased from Thermo Fisher Scientific, U.S.A. PEI MAX- transfection agent from Polysciences. Mouse monoclonal anti-α-tubulin was purchased from Sigma (Cat # T5168); Mouse monoclonal anti-γ-tubulin for WB & IF (Cat # sc-17787), mouse monoclonal anti-TACC3 (Cat # sc-376883), mouse polyclonal anti-GCP4 (Cat # sc-271876), mouse monoclonal anti-GCP3 (Cat # sc-373758), anti-GCP6 (Cat # sc-374063), and mouse monoclonal anti-GFP antibody (Cat # sc-9996) were obtained from Santa Cruz Biotechnology, Inc.; rabbit polyclonal anti-ch-TOG antibody was obtained from Abcam (Cat # ab-236981); mouse monoclonal anti-actin was purchased from BD Biosciences (Cat # 612656); and rabbit monoclonal phospho-TACC3 antibody was purchased from Cell Signalling, U.S.A. (Cat # 8842). The secondary antibodies, anti-rabbit Alexa 568 and anti-mouse Cy5 were obtained from Jackson ImmunoResearch. DAPI was purchased from Sigma. GFP-Trap agarose beads used for IP were from Chromotech. Agarose A/G beads used for IP were from Santa Cruz Biotechnology, Inc.