En3hance

To analyze newly synthesized RNA, cells were pulse‐labeled for 2 h with 1.2 μCi/ml of [³H]‐uridine (PerkinElmer) and then chased in non‐radioactive media for 4 h before TRIzol RNA extraction as described above. 2 μg of total RNA were resolved either on a formaldehyde‐containing 1.2% agarose gel for 18S and 28S rRNAs or on a TBE‐urea 10% polyacrylamide gel for 5S rRNA, 5.8S rRNA, or tRNAs and transferred to Hybond N+ membrane (GE Healthcare). After ultraviolet cross‐linking, the membranes were sprayed with EN3HANCE (PerkinElmer) and exposed to Kodak BioMax MS film (Kodak) at −80°C for 1 week.

The HMT assay was performed as described in Iglesias et al., 2018 (link). Briefly, the [3H]-based HMT assay was carried out in a 10 µl reaction mix containing 2 µM histone or nucleosome, 5.6 µM [3H]- S-adenosyl methionine (SAM), 0.3–1 µM enzyme, and 1x HMT buffer (50 mM Tris-HCl, pH 8.0, 20 mM KCl, 10 mM MgCl₂, 0.02% Triton X-100, 1 mM DTT, 5% glycerol, and 1 mM PMSF). The reaction mix was incubated at 20°C for 2 hr, and then loaded onto 17% SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membrane, which was then soaked with autoradiography enhancer (EN3HANCE, PerkinElmer) and then air dried. Fluorography signal was detected by X-ray film. For the histone mass spectrometry analysis, 150 µl reaction mix containing approximately 0.3 µM GST-SET-32, 2.5 µM H3, and 213 µM SAM and 1 x HMT buffer without Triton X-100 was used.

In vitro methylation assay was performed as described with minor modification (Hartsough et al., 2013 (link)). Briefly, VEGFR-2 was immunoprecipitated from HEK-293 cells expressing VEGFR-2 alone or co-expressing VEGFR-2 and PRMT4 were incubated with the methylation reaction buffer ((50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl2, 1 mM β-mercaptoethanol, and 100 mM sucrose) plus 1 μL of adenosyl-L-methionine, S-[methyl-3H] (1 mCi/mL stock solution, Perkin Elmer) at 30°C. In some experiments, a purified GST-PRMT4 also added into the reaction mix. The reaction stopped by adding 2× SDS loading buffer after 1 h and was resolved on SDS-PAGE. The separated protein samples were then transferred from the gel to a polyvinylidene difluoride (PVDF) membrane, sprayed with EN3HANCE (Perkin Elmer) and exposed to X-ray film.

1-beta-D-arabinofuranosylcytosine (AraC) and benzidine were obtained from Sigma-Aldrich. S-adenosyl-L-[methyl-³H] methionine (³H-AdoMet, 0.55 mCi/ml, NET-155H) and fluorographic enhancer, EN³HANCE, were from PerkinElmer. The pFlag-CMV2-p38α plasmid [3 (link)] was used as a template for PCR to generate mutations on R49 and R149. These plasmids were subsequently cloned into the pET6H vector for expression of recombinant proteins.

Reaction products and standards were dissolved in 20% 1-propanol and separated on a 10 cm HPTLC Si-60 plates (Merck) using 1-propanol:acetone:water 9:6:4 (v:v:v) as mobile phase. Non-radiolabelled sugars were visualized by orcinol/H₂SO₄ staining. In the case of radiolabelled products, the HPTLC plates were sprayed with En³hance (PerkinElmer) and visualized by fluorography.

In vitro methylation reactions were performed in 30 μl PBS (pH 7.4) with 1 μg of peptide, 1 μg of recombinant enzymes (PRMT1, CARM1 or PRMT9) and 0.42 μM S-adenosyl-l-[methyl-³H]methionine (Perkin Elmer). Reactions were incubated at 30 °C for 1 hour, resolved on 15% SDS-PAGE, transferred to PVDF membrane, treated with En³Hance (Perkin Elmer) and exposed to film at −80 °C. Exposure time was different for each PRMT: PRMT1 for 3 days, CARM1 for 2 days, and PRMT9 for 12 days.

Fusions of JMJD2A aa or SET7/9 with GST were produced in E. coli and affinity purified with the help of glutathione agarose beads (71 (link)). The 6His/Flag-tagged JMJD2A was expressed with the help of a baculovirus expression system and purified on Ni²⁺-NTA agarose (13 (link)). These recombinant proteins (1–3 μg) were incubated for 2 hours at 30°C in the presence of 1 μM ³H-SAM (60 Ci/mmol) in 50 mM Tris-HCl (pH 8.5), 5 mM MgCl₂, and 4 mM DTT. Samples were then subjected to SDS-PAGE, transferred to PVDF membrane, and visualized with Ponceau S staining (72 (link)). The dried membrane was sprayed with EN³HANCE (PerkinElmer) 4 times, waiting for 10 minutes between each application. After a final drying for 30 minutes, membranes were exposed to film at –80°C without intensifying screen.