Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation decreases methyltransferase activity and intrinsic protein stability of SETD2 catalytic core in vitro . A , upper panel : schematic representation of the SETD2 domains. The SETD2 L1609P mutation is located in the SET domain within the SETD2 catalytic core (composed of the AWS, SET, and post-SET domains). Lower left panel : Structural representation of the SETD2 active site (PDB entry: 5JJY ) with a zoomed-in view of the substrate (H3K36M peptide) and cofactor (SAH) binding sites. Lower right panel : Sequence alignment of residues 1603 to 1619 of the SET domain of human SETD2 with the equivalent sequences of human G9A, EZH2, NSD1, NSD2, SETD8, MLL1, MLL2, SETD8, ASH1 (sequence retrieved from the UniProt database). Conserved residues are highlighted in blue . The secondary structure of the SETD2 residues (deduced from PDB entry: 5JJY ) is shown above the alignment. The SETD2 residue L1609 and the equivalent residues in the other SET domain-containing enzymes are highlighted in orange . B , in vitro methylation of recombinant histone H3, core histones (purified from HEK293T SETD2-KO cells) or recombinant nucleosomes. SETD2-dependent H3K36me3 methylation was detected using an anti-H3K36me3 antibody. Ponceau Red staining of histones is shown. The purified catalytic core of SETD2 WT and SETD2 L1609P mutant used in the assays were detected using an anti-6xHis-tag antibody. C , SETD2 mono-methylation, dimethylation, or trimethylation activities were determined by UFLC assays using H3K36 fluorescent peptides as previously described ( , ). Bar graphs and error bars represent the mean and SD of three independent experiments. D , automethylation of SETD2 and methylation of α-tubulin detected by autoradiography using 3 H-SAM. Coomassie Blue staining was used as loading control. E , determination of the intrinsic protein stability of SETD2 WT or SETD2 L1609P by thermal shift assay (TSA). Left panel : T m values were determined by the minimum of the first derivative of the fluorescence emission as a function of temperature (dFluo/dT). Right panel : Bar graphs and error bars represent the mean and SD of nine experiments. SETD2, SET-domain containing protein 2; UFLC, ultrafast liquid chromatography.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Activity Assay, In Vitro, Binding Assay, Sequencing, Residue, Methylation, Recombinant, Purification, Staining, Autoradiography, Control, Thermal Shift Assay, Fluorescence, Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation results in low levels of the H3K36me3 mark and in low expression of SETD2 in CRISPR/Cas9-engineered HEK293T cells and in transfected HEK293T-SETD2 KO cells . A , endogenous H3K36me3 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : the H3K36me3 mark was detected by immunofluorescence using an anti-H3K36me3 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with 10 μm scale bars. Right panel : Histones from CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant were extracted and H3K36me3 levels were determined by Western blotting using a an anti-H3K36me3 antibody. Ponceau Red staining of extracted histones is shown. B , endogenous SETD2 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : Cells were fixed and SETD2 was detected using an anti-SETD2 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with scale bars of 10 μm. Right panel : SETD2 was detected in cell extracts by Western blot using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. C , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 L1609P were transfected with GFP-SETD2 WT or GFP-SETD2 L1609P plasmids. Nontransfected CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were used as controls. Ectopic GFP-SETD2 expression and H3K36me3 mark levels were detected by Western blot using anti-GFP or anti-H3K36me3 antibodies, respectively. Ponceau Red staining of cellular histones or extracts on membranes are shown. D , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were treated with MG132 or DMSO. Endogenous SETD2 WT and SETD2 L1609P expression levels were detected by Western blotting using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Expressing, CRISPR, Transfection, Immunofluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Overall structure of the ternary complex of SETD2 L1609P mutant bound to H3K36M peptide and SAM cofactor . A , left panel : cartoon representation of SETD2 WT (PDB: 5JJY ) ( cyan ) bound to H3K36M peptide ( orange ) and the SAH cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 L1609 and H3M36 residues are represented by yellow and orange sticks , respectively. The close-up view shows the region around residue L1609 with the H3K36M peptide (residues 29–42, orange ) and the SAH cofactor ( black sticks ). Zinc atoms are shown in gray . Right panel : cartoon representation of the SETD2 L1609P mutant (PDB: 8RZU ) ( salmon ) bound to the H3K36M peptide ( green ) and the SAM cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 P1609 and H3M36 residues are shown as yellow and green sticks , respectively. The close-up view shows the region around the residue P1609 with the H3K36M peptide (residues 29–39, green ) and the SAM cofactor ( black sticks ). B , left panel : cartoon representation of the characteristic triangular shape of the SET domain formed by 3 β-sheets (β1-β2; β3-β8-β7; β4-β6-β5 strands) of SETD2 WT in complex with the H3K36M peptide (residues 29–42 in orange) (PDB: 5JJY ). The β-sheet composed of β4-β6-β5 strands is boxed and the SETD2 L1609 residue is shown in yellow . Right panel : cartoon representation of the triangular β-sheet structure of the SET domain of the SETD2 L1609P mutant ( salmon ) in complex with the H3K36M peptide (residues 29–39, green ) (PDB: 8RZU ). The β5-strand in SETD2 WT adopts a loop conformation in the structure of the SETD2 L1609P mutant ( boxed ). The P1609 residue in mutant SETD2 is shown in yellow . SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Effects of the SETD2 L1609P mutation on the conformations of neighboring residues of SETD2 and the H3K36M peptide. A , the left panel shows a cartoon overlay of the β5-β6 hairpin of SETD2 WT (PDB: 5JJY ) ( cyan ) and SETD2 L1609P mutant ( salmon ) structures. The H3K36M peptide is shown in orange and green for SETD2 WT and SETD2 L1609P, respectively. The side chains of residues L1609 and P1609 residues are shown as sticks ( yellow CPK). The middle panel shows a close-up view of the hairpin residues (1609–1613) of SETD2 WT ( cyan ) and SETD2 L1609P ( salmon ). The side chains are shown in CPK sticks . The right panel shows the β5-β6 hairpin residues of SETD2 WT ( top ) and SETD2 L1609P ( bottom ) in sticks . Dashes represent the distance between Cα of residues K1610 and E1613 residues. B , conformational remodeling of residues K1610 and K1639 of SETD2 and residue K37 of H3 induced by the L1609P mutation. Left panel shows residues SETD2 L1609 ( yellow ), K1610 (cyan), K1639 ( cyan ), and H3K37 ( orange ) in spheres and sticks in the SETD2 WT structure (PDB: 5JJY ). Middle panel shows residues SETD2 P1609 ( yellow ), K1610 ( salmon ), K1639 ( salmon ), and H3K37 ( green ) in spheres and sticks in the SETD2 L1609P structure. The right panel shows residues P1609 ( yellow ) and K1610 ( salmon ) from the SETD2 L1609P structure and residues K1639 ( cyan ) and H3K37 ( orange ) from the SETD2 WT structure. Steric clashes between side chains are shown in boxes . The orientations are the same in all three panels and were obtained by superimposing the SETD2 WT and L1609P main chains. C , surface representation of the SETD2 substrate-binding region. H3K36M peptides are shown as sticks. The left panel shows the SETD2 WT structure (PDB: 5JJY ) in light cyan . The SETD2 L1609 residue is shown in yellow . The SETD2 K1610 and K1639 residues are shown in blue . H3K36M peptide residues diffracting in both WT and L1609P structures (residues A29–H39) are shown in green . H3K36M peptide residues observed only in the SETD2 WT structure (residues R40-R42) are shown in transparent orange . The right panel shows the SETD2 L1609P structure in light pink . The SETD2 P1609 residue is shown in yellow . The K1610 and K1639 residues are shown in purple . H3K36M peptide residues observed in the SETD2 L1609P structure (A29–H39) are shown in green . SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Details of H3K36M peptide recognition by SETD2 L1609P mutant . A , the left panel shows a clipped surface representation of the SETD2 WT-H3K36M peptide complex (PDB: 5JJY ). Peptide residues (residues A29–R42) are represented by sticks . The right panel shows a clipped surface representation of the SETD2 L1609P-H3K36M peptide complex. Peptide residues (A29–H39) are represented by sticks . The structures of the SETD2-H3K36M peptide complexes are shown in the same orientation after superimposition of the main chains. B , upper panel : Structural alignment of H3K36M peptides (residues A29–H39) in SETD2 WT (PDB: 5JJY ) ( orange ) and SETD2 L1609P ( green ) structures. Lower panel : Differences between SETD2-H3K36M peptide interactions in SETD2 WT and SETD2 L1609P complexes. Residue interactions across the binding interface of SETD2 WT or SETD2 L1609P mutant with H3K36M peptide were determined using LIGPLOT . Residues are represented by sticks . Residues involved in SETD2-H3K36M peptide interactions (nonbonded and hydrogen bonds) are represented by sticks and spheres . Dashes represent hydrogen bond. The lower left panel shows the SETD2 WT ( cyan )-H3M36 ( orange ) interacting residues that are specific for the SETD2 WT complex and not present in the SETD2 L1609P complex. These interactions are listed in a table ( bottom left ). The lower right panel shows SETD2 L1609P ( salmon )-H3K36M ( green ) peptide interacting residues that are specific for the SETD2 L1609P complex and not present in the SETD2 WT complex. These interactions are listed in a table ( bottom right ). SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Residue, Binding Assay

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 1. Organoselenium compound ebselen is an inhibitor of the YTH domain. (A) Binding specificity of the YTH domain toward m6A compared to adenosine. Data were fitted using a three-parameter nonlinear regression model R2 = 0.9870, EC50 = 596 μM. NFUs = normalized fluorescence units. (B) Fluorescence quenching by the ssRNA containing the GGm6ACU variant compared to the unmethylated one. Data were fitted using a three- parameter nonlinear regression model. R2 = 0.9386, EC50 = 1.05 μM. NFUs = normalized fluorescence units. (C) Fluorescence quenching by the ssRNA containing the GAm6ACU consensus variant compared to the unmethylated one. Data were fitted using a three-parameter nonlinear regression model. R2 = 0.9484, EC50 = 0.139 μM. NFUs = normalized fluorescence units. (D) Plot of progressive Z-score values of fluorescence intensity (FI) of 2560 compounds according to their quenching effect on the YTHDF1 domain. (E) Molecular structure of ebselen (1), the selected hit compound.

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques: Binding Assay, Fluorescence, Variant Assay

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 2. Organoselenium compound ebselen is an inhibitor of the YTH domain and can bind YTHDF1 in cells. (A) Dose−response curves of increasing amounts of ebselen added to the YTHDF1 and YTHDF2 protein domains were obtained with the tryptophan quenching assay. Data were fitted with a four-parameter nonlinear regression model, R2 = 0.954 and 0.93, EC50 of 1.63 and 1.66 μM for the YTHDF1 and YTHDF2 YTH domains, respectively. (B) Ebselen cannot reduce tryptophan fluorescence of the YTH domain of the YTHDC1 protein. (C) Dynamic mass redistribution (DMR) assay to evaluate ebselen binding at equilibrium. Measurements were performed before (baseline) and after (final) compound addition. The response (in picometers (pm)) was measured by subtracting the baseline output from the final output signals. The output signal for each well was obtained by subtracting the signal of the protein-coated reference area from the signal of the uncoated area. The data were fitted to a sigmoidal function

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques: Fluorescence, Binding Assay

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 3. Ebselen affects the viability of prostate cancer cells and interferes with the RNA-binding ability of YTHDF2. (A) Cell viability was determined with an OZblue kit after 24, 48, and 72 h of treatment with different ebselen concentrations (1, 5, 10, 25, 50, 100, and 200 μM). Data were normalized and fitted with a four-parameter nonlinear model (24 h: R2 = 0.9632, IC50 = 58.77 μM; 48 h: R2 = 0.9133, IC50 = 49.58 μM; and 72 h:R2 = 0.9687, IC50 = 26.83 μM). (B) m6A levels in poly(A) purified mRNA were quantified by two-dimensional (2D) thin-layer chromatography (TLC, see the Materials and Methods section). The quantification is shown. n = 2 independent experiments; error bars, s.e. (C) Diagram showing the selected targets derived from the intersection of three different PAR-CLIP datasets on GEO (GSE63591 for YTHDF1, GSE49339 for YTHDF2, and

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques: RNA Binding Assay, Purification, Thin Layer Chromatography, Derivative Assay

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 4. Ebselen covalently binds to the YTH domain of the YTHDF1 protein, and the substitution of the Se atom with S maintains its ability to interact with the domain and disrupt its RNA-binding ability. (A) Dose−response curve obtained with increasing concentrations of ebsulfur in the tryptophan quenching assay. Data were fitted with a four-parameter nonlinear regression model, R2 = 0.924 and EC50 = 1.76 ± 0.02 μM. (B) Determination of the IC50 value of the ebsulfur molecule with the AlphaScreen assay, using nonlinear regression fits of the data according to a four- parameter nonlinear regression model: R2 = 0.915, IC50 = 23.14 ± 0.08 μM. (C, D) Compound 3 failed to generate a dose−response curve in the tryptophan quenching assay and to disrupt the RNA-binding ability of the YTH domain in the AlphaScreen assay. (E) Mass spectra of the YTH domain of YTHDF1 (4RCJ structure in PDB), alone or in the presence of ebselen (50 μM) with or without 1 mM DTT.

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques: RNA Binding Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 5. Ebselen interferes with the correct organization of the m6A-binding pocket. (A, B) Ebselen binds the YTHDF1 YTH domain adopting different poses; the protein matrix is shown in green with ebselen in cyan for pose 1, while for binding mode 2, the YTH domain is in violet with the ebselen molecule in slate. The selenium anomalous map is contoured at 3.5σ and shown in orange. (C) In the holo 4RCJ structure (dark yellow), the β4−β5 loop organizes its structure on the bound m6A (yellow); in the apo 4RCI structure (lime), the loop is disordered, but Trp470 inserts into the binding pocket; and ebselen (color code: cyan for pose 1, slate for pose 2) binding is incompatible with the m6A-binding-competent conformation. (D) Due to Trp465 or Trp470 displacement, ebselen enlarges the m6A pocket; the druggable pocket has been identified with DoGSiteScorer.46

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques: Binding Assay

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 6. Ebselen specifically interacts with the hydrophobic pocket of the YTH domain. (A) Plot of the decreases in signal intensity of the YTHDF1 YTH domain (100 μM) in the presence of the ligand ebselen (50 μM); the residues exhibiting the most significant decreases are highlighted in red. The stars indicate residues with a significant decrease in signal intensity but overlapping in the NMR spectra. (B) Plot of the chemical shift perturbations (CSPs) of the YTHDF1 YTH domain (100 μM) in the presence of the ligand ebselen (50 μM), evaluated according to the formula

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques:

Journal: ACS pharmacology & translational science

Article Title: Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6 -Methyladenosine-Modified RNAs.

doi: 10.1021/acsptsci.2c00008

Figure Lengend Snippet: Figure 7. Molecular structure of new synthetic ebselen and ebsulfur analogues and co-crystal structures of compounds 7 and 9 with the YTHDF1 YTH domain. (A) Molecular structures of ebselen and ebsulfur analogues. (B) Compound 7 (magenta) and 9 (green) interact with the YTH domain through a disulfide bond with Cys412 but are oppositely directed with respect to ebselen (violet). (C, D) Detailed interaction of compound 7 and 9 with the YTH protein matrix.

Article Snippet: The human YTHDF1 YTH domain (amino acids 365−554, PDB: 4RCJ), (Addgene plasmid # 64654, plasmid pET28a-MHL, 6× His Tag at the Cterminal), and the human YTHDF2 YTH domain (amino acids 383−553, PDB: 4WQN, cloned in pET21b(+) 6× His Tag at the N-terminal) were expressed in the BL21(DE3) Escherichia coli strain cultured in a Luria−Bertani medium at 37 °C till OD= 0.6−0.8 and then shifted at 18 °C O/N after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Techniques: Analogues