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EpiCypher recombinant setd2

The <t>L1609P</t> mutation decreases methyltransferase activity and intrinsic protein stability of <t>SETD2</t> 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.

Recombinant Setd2, supplied by EpiCypher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+setd2/SETD2%2C+Recombinant+Human/pmc12969715-275-3-32
Average 94 stars, based on 2 article reviews

recombinant setd2 - by Bioz Stars, 2026-10

94/100 stars

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1) Product Images from "The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation"

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

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2026.111259

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Mutagenesis, Residue

$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.$
Figure Legend 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.

Techniques Used: Mutagenesis, Residue, Binding Assay

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.

Figure Legend 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.

Techniques Used: Mutagenesis, Residue, Binding Assay

Activity Assay:

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation.
Article Snippet: .. Jo ur al Pr e-p roo f 15 Determination of SETD2 H3K36-methyltransferase activity using recombinant H3 histone, HEK293T SETD2 KO purified core-histones or recombinant nucleosome substrates Five μM of recombinant SETD2 (WT, L1609P or T1663M) were incubated overnight at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16-0009, Epicypher) or 2 μg core histones purified from HEK293T SETD2 KO cells (35), 100 μM SAM and 1 mM DTT. ..

Recombinant:

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation
Article Snippet: .. Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT. ..

Purification:

Incubation:

Image Search Results

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: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

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.

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.

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.

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.

Techniques: Mutagenesis, Residue, Binding Assay

$( A ) Immunoblot analysis showing localization of SETD2 in whole-cell extracts (WCE) and both nuclear and cytoplasmic (Cyto) compartments of 786-0 and HEK293T cells. Lamin A/C and lactate dehydrogenase (LDH) are used as controls for the nuclear and cytoplasmic fractions, respectively. ( B ) Immunoblot analysis showing coimmunoprecipitation of endogenous SETD2 and endogenous actin in 786-0 cells using SETD2 antibodies from two different sources. ( C ) Immunoblot analysis showing that actin is methylated in SETD2-proficient but not in SETD2-deficient 786-0 cells by immunoprecipitation (IP) of endogenous actin using two different antibodies directed against the SETD2 trimethyl-lysine epitope. Data in (A to C) are representative of experiments repeated at least three times with similar results. ( D ) Deconvolution microscopy imaging of filamentous (F)–actin using phalloidin (green) and SETD2 methyl epitope–specific antibody (magenta) showing colocalization of the methyl mark with F-actin in 786-0 cells. Scale bars, 10 μm. ( E ) Representative intensity profiles of the staining observed with the methyl-specific antibody and phalloidin. Merged images from (D) are shown on the left to indicate the position for line profiles. ( F ) Quantification of intensity profiles seen in (E). y axis represents the ratio of methyl antibody to phalloidin intensity. Each small circle represents a single cell. Large circles represent means from 20 cells for each independent biological replicate; data are means ± SEM from these values ( n = 3).$

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Immunoblot analysis showing localization of SETD2 in whole-cell extracts (WCE) and both nuclear and cytoplasmic (Cyto) compartments of 786-0 and HEK293T cells. Lamin A/C and lactate dehydrogenase (LDH) are used as controls for the nuclear and cytoplasmic fractions, respectively. ( B ) Immunoblot analysis showing coimmunoprecipitation of endogenous SETD2 and endogenous actin in 786-0 cells using SETD2 antibodies from two different sources. ( C ) Immunoblot analysis showing that actin is methylated in SETD2-proficient but not in SETD2-deficient 786-0 cells by immunoprecipitation (IP) of endogenous actin using two different antibodies directed against the SETD2 trimethyl-lysine epitope. Data in (A to C) are representative of experiments repeated at least three times with similar results. ( D ) Deconvolution microscopy imaging of filamentous (F)–actin using phalloidin (green) and SETD2 methyl epitope–specific antibody (magenta) showing colocalization of the methyl mark with F-actin in 786-0 cells. Scale bars, 10 μm. ( E ) Representative intensity profiles of the staining observed with the methyl-specific antibody and phalloidin. Merged images from (D) are shown on the left to indicate the position for line profiles. ( F ) Quantification of intensity profiles seen in (E). y axis represents the ratio of methyl antibody to phalloidin intensity. Each small circle represents a single cell. Large circles represent means from 20 cells for each independent biological replicate; data are means ± SEM from these values ( n = 3).

Article Snippet: The following commercially available proteins were used in this study: recombinant SETD2 (amino acids 1418 to 1714) protein (Active Motif, 31358), rabbit skeletal muscle actin (Cytoskeleton, Inc., AKL95), bovine cardiac muscle actin (Cytoskeleton, Inc., AD99), chicken gizzard smooth muscle actin (Cytoskeleton, Inc., AS99), β-actin (NM_001101) human recombinant protein (OriGene, TP303643), and β-actin (NM_001101) human recombinant protein (OriGene, TP720518).

Techniques: Western Blot, Methylation, Immunoprecipitation, Microscopy, Imaging, Staining

( A ) Autoradiography showing in vitro methylation of actin using tritiated S -adenosylmethionine ( 3 H-SAM) as methyl group donor and recombinant glutathione- S -transferase (GST)–tagged SETD2 catalytic SET domain (amino acids 1418 to 1714). Film shows automethylation of SETD2 and histone methylation as positive control. Data are representative of experiments repeated at least three times, with similar results. ( B ) Fluorescence-based in vitro methylation using recombinant tSETD2 (amino acids 1418 to 2564) with purified cardiac muscle (red), smooth muscle (green), skeletal muscle (purple) actins, and recombinant actin (rActin) from HEK293T cells (blue) or E. coli (orange). Data are means ± SEM ( n = 2). ( C ) Fluorescence-based in vitro methylation of actin using recombinant tSETD2 (amino acids 1418 to 2564) following addition of Latrunculin A (LatA) at indicated concentrations. Methyltransferase activity calculated by slope of linear regression from fluorescence trace (a.u./min), against LatA concentration on x axis. Data are means ± SEM ( n = 3). y axis in (B) and (C) plotted after subtracting automethylation from samples with SETD2 alone. ( D and E ) Immunoblot analysis showing recognition of actin proteins by SETD2 methyl epitope antibodies anti-Me3 K40 (D) and anti-Me3 K36 (E) following in vitro methylation with recombinant GST-tagged SETD2 (amino acids 1418 to 1714). Data are representative of experiments repeated at least three times with similar results.

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Autoradiography showing in vitro methylation of actin using tritiated S -adenosylmethionine ( 3 H-SAM) as methyl group donor and recombinant glutathione- S -transferase (GST)–tagged SETD2 catalytic SET domain (amino acids 1418 to 1714). Film shows automethylation of SETD2 and histone methylation as positive control. Data are representative of experiments repeated at least three times, with similar results. ( B ) Fluorescence-based in vitro methylation using recombinant tSETD2 (amino acids 1418 to 2564) with purified cardiac muscle (red), smooth muscle (green), skeletal muscle (purple) actins, and recombinant actin (rActin) from HEK293T cells (blue) or E. coli (orange). Data are means ± SEM ( n = 2). ( C ) Fluorescence-based in vitro methylation of actin using recombinant tSETD2 (amino acids 1418 to 2564) following addition of Latrunculin A (LatA) at indicated concentrations. Methyltransferase activity calculated by slope of linear regression from fluorescence trace (a.u./min), against LatA concentration on x axis. Data are means ± SEM ( n = 3). y axis in (B) and (C) plotted after subtracting automethylation from samples with SETD2 alone. ( D and E ) Immunoblot analysis showing recognition of actin proteins by SETD2 methyl epitope antibodies anti-Me3 K40 (D) and anti-Me3 K36 (E) following in vitro methylation with recombinant GST-tagged SETD2 (amino acids 1418 to 1714). Data are representative of experiments repeated at least three times with similar results.

Techniques: Autoradiography, In Vitro, Methylation, Recombinant, Positive Control, Fluorescence, Purification, Activity Assay, Concentration Assay, Western Blot

( A ) Amino acid sequence showing conserved KxP SETD2 recognition motif present in all actin isoforms. Position of the lysine residue varies depending on actin isoform; reference to this site as “ActK68” is based on its position in β-actin (ACTB). Histone H3 sequence containing the KxP motif is shown below for reference. ( B ) Representative tandem mass spectrometry (MS/MS) spectrum of trimethylated ActK68 peptides recovered from SETD2-proficient 786-0 cells. m / z , mass/charge ratio. ( C ) Fluorescence-based in vitro methylation assay showing in vitro methylation of biotin-labeled K68-containing actin peptides (amino acids 62 to 78) with recombinant tSETD2 (amino acids 1418 to 2564). Sequence for the peptides used is shown in (A). Data are means ± SEM ( n = 4). ( D ) Immunoblot analysis showing dependency of the ActK68me3 mark on SETD2 by IP of endogenous actin from whole-cell extracts of SETD2-proficient and SETD2-deficient 786-0 cells using the anti-Me3 K68 antibody. Data are representative of experiments repeated at least three times with similar results.

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Amino acid sequence showing conserved KxP SETD2 recognition motif present in all actin isoforms. Position of the lysine residue varies depending on actin isoform; reference to this site as “ActK68” is based on its position in β-actin (ACTB). Histone H3 sequence containing the KxP motif is shown below for reference. ( B ) Representative tandem mass spectrometry (MS/MS) spectrum of trimethylated ActK68 peptides recovered from SETD2-proficient 786-0 cells. m / z , mass/charge ratio. ( C ) Fluorescence-based in vitro methylation assay showing in vitro methylation of biotin-labeled K68-containing actin peptides (amino acids 62 to 78) with recombinant tSETD2 (amino acids 1418 to 2564). Sequence for the peptides used is shown in (A). Data are means ± SEM ( n = 4). ( D ) Immunoblot analysis showing dependency of the ActK68me3 mark on SETD2 by IP of endogenous actin from whole-cell extracts of SETD2-proficient and SETD2-deficient 786-0 cells using the anti-Me3 K68 antibody. Data are representative of experiments repeated at least three times with similar results.

Techniques: Sequencing, Residue, Mass Spectrometry, Tandem Mass Spectroscopy, Fluorescence, In Vitro, Methylation, Labeling, Recombinant, Western Blot

( A ) Immunoblot analysis showing decreased F-actin in SETD2-deficient 786-0, HEK293T, and MEF cells. Whole-cell lysate shows absence of SETD2, associated with the expected loss of histone H3K36me3 methylation. ( B ) Quantitation of F-/G- actin ratio (top) and whole-cell lysate actin (bottom) from the data is shown in (A). Data are means ± SEM ( n = 3 for 786-0 and HEK293T; n = 6 for MEF). ( C ) Immunoblot analysis of F-/G-actin ratio in HEK293T cells expressing wild-type or K68A/R mCherry–β-actin. ( D ) Immunoblot analysis of whole-cell lysates shows no change in total actin levels or SETD2 histone methylation with expression of K68A/R mCherry–β-actin. ( E ) Quantitation of F-/G-actin and total actin shown in (C) and (D), respectively. Data are means ± SEM ( n = 3).

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Immunoblot analysis showing decreased F-actin in SETD2-deficient 786-0, HEK293T, and MEF cells. Whole-cell lysate shows absence of SETD2, associated with the expected loss of histone H3K36me3 methylation. ( B ) Quantitation of F-/G- actin ratio (top) and whole-cell lysate actin (bottom) from the data is shown in (A). Data are means ± SEM ( n = 3 for 786-0 and HEK293T; n = 6 for MEF). ( C ) Immunoblot analysis of F-/G-actin ratio in HEK293T cells expressing wild-type or K68A/R mCherry–β-actin. ( D ) Immunoblot analysis of whole-cell lysates shows no change in total actin levels or SETD2 histone methylation with expression of K68A/R mCherry–β-actin. ( E ) Quantitation of F-/G-actin and total actin shown in (C) and (D), respectively. Data are means ± SEM ( n = 3).

Techniques: Western Blot, Methylation, Quantitation Assay, Expressing

$Immunoblot (IB) analysis ( A ) and quantitation ( B ) showing changes in actin polymerization (F-actin in the insoluble fraction) following washout after treatment with the actin depolymerizing agent latrunculin A (LatA). Data are means ± SEM ( n = 4). ( C ) IB analysis using anti-Me3 K68 antibody showing that ActK68me3 occurs on endogenous actin from the insoluble F-actin fraction of SETD2-proficient 786-0 cells; tubulin (which is also methylated by SETD2) is not found in this fraction. Data are representative of experiments repeated at least three times with similar results. Absence of H3K36me3 in the insoluble fraction is used as a control to confirm loss of SETD2 in (A) and (C). ( D ) IP of actin using anti-Me3 K68 antibody from soluble (G-actin) and insoluble (F-actin) fractions of SETD2-proficient cells treated with LatA for indicated times. Light and dark exposures are shown for contrast in amount of methylated actin between fractions. Data are representative of three experiments performed using anti-Me3 K68 and anti-Me3 Pan antibodies with similar results. In an additional experiment performed shortly after cells were brought up from cryopreservation, we saw a less marked loss of methylated insoluble actin with LatA, but saw no increase in methylated soluble actin, consistent with data shown here.$

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: Immunoblot (IB) analysis ( A ) and quantitation ( B ) showing changes in actin polymerization (F-actin in the insoluble fraction) following washout after treatment with the actin depolymerizing agent latrunculin A (LatA). Data are means ± SEM ( n = 4). ( C ) IB analysis using anti-Me3 K68 antibody showing that ActK68me3 occurs on endogenous actin from the insoluble F-actin fraction of SETD2-proficient 786-0 cells; tubulin (which is also methylated by SETD2) is not found in this fraction. Data are representative of experiments repeated at least three times with similar results. Absence of H3K36me3 in the insoluble fraction is used as a control to confirm loss of SETD2 in (A) and (C). ( D ) IP of actin using anti-Me3 K68 antibody from soluble (G-actin) and insoluble (F-actin) fractions of SETD2-proficient cells treated with LatA for indicated times. Light and dark exposures are shown for contrast in amount of methylated actin between fractions. Data are representative of three experiments performed using anti-Me3 K68 and anti-Me3 Pan antibodies with similar results. In an additional experiment performed shortly after cells were brought up from cryopreservation, we saw a less marked loss of methylated insoluble actin with LatA, but saw no increase in methylated soluble actin, consistent with data shown here.

Techniques: Western Blot, Quantitation Assay, Methylation, Control

( A ) In vitro scratch assay at 0 and 24 hours after wound inflection, illustrating that SETD2-deficient cells migrate slower than SETD2-proficient 786-0 cells. Scale bars, 1000 μm. ( B ) Quantification of scratch assays seen in (A). Small circles represent each independent measurement across all experiments. Large circles represent mean from a minimum of nine individual measurements for each independent biological replicate ( n = 4).

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) In vitro scratch assay at 0 and 24 hours after wound inflection, illustrating that SETD2-deficient cells migrate slower than SETD2-proficient 786-0 cells. Scale bars, 1000 μm. ( B ) Quantification of scratch assays seen in (A). Small circles represent each independent measurement across all experiments. Large circles represent mean from a minimum of nine individual measurements for each independent biological replicate ( n = 4).

Techniques: In Vitro, Wound Healing Assay

( A ) Schematic diagram showing interaction between the SETD2 C-terminal and HTT N-terminal proline-rich region (PRR), as reported in ( , ). ( B ) Immunoblot (IB) analysis showing coimmunoprecipitation of mCherry-tagged SETD2 C-terminal [SETD2-(C)-mCherry] and endogenous HTT in HEK293T cells. ( C ) IB analysis showing that actin methylation by SETD2 is dependent on both HTT and HIP1R using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) to immunoprecipitate actin from 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Input lysates shown to confirm knockdown of HTT and HIP1R. ( D and E ) IB analysis (D) and quantitation (E) of decreased insoluble F-actin in 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Data are means ± SEM ( n = 3). ( F ) Quantitation of migration data after siRNA-mediated knockdown of HTT or HIP1R in SETD2-proficient versus SETD2-deficient 786-0 cells. Small circles each represent an independent measurement from all biological replicates. Large circles represent mean from 12 measurements for each independent biological replicate ( n = 4). ( G ) IB analysis showing decreased actin methylation after expression of a mutant HTT protein by IP of actin using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) from HEK293T cells expressing a doxycycline (Doxy)–inducible CFP-tagged N-terminal HTT construct containing 94 polyglutamine repeats (CFP-HTT94Q) and a retrotransactivator (rtTA3). Input lysate confirms expression of CFP-tagged mutant protein and loss of SETD2 methylation activity (H3K36me3) without change in endogenous SETD2 expression. Data in (B), (C), and (G) are representative of experiments repeated three times with similar results.

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Schematic diagram showing interaction between the SETD2 C-terminal and HTT N-terminal proline-rich region (PRR), as reported in ( , ). ( B ) Immunoblot (IB) analysis showing coimmunoprecipitation of mCherry-tagged SETD2 C-terminal [SETD2-(C)-mCherry] and endogenous HTT in HEK293T cells. ( C ) IB analysis showing that actin methylation by SETD2 is dependent on both HTT and HIP1R using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) to immunoprecipitate actin from 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Input lysates shown to confirm knockdown of HTT and HIP1R. ( D and E ) IB analysis (D) and quantitation (E) of decreased insoluble F-actin in 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Data are means ± SEM ( n = 3). ( F ) Quantitation of migration data after siRNA-mediated knockdown of HTT or HIP1R in SETD2-proficient versus SETD2-deficient 786-0 cells. Small circles each represent an independent measurement from all biological replicates. Large circles represent mean from 12 measurements for each independent biological replicate ( n = 4). ( G ) IB analysis showing decreased actin methylation after expression of a mutant HTT protein by IP of actin using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) from HEK293T cells expressing a doxycycline (Doxy)–inducible CFP-tagged N-terminal HTT construct containing 94 polyglutamine repeats (CFP-HTT94Q) and a retrotransactivator (rtTA3). Input lysate confirms expression of CFP-tagged mutant protein and loss of SETD2 methylation activity (H3K36me3) without change in endogenous SETD2 expression. Data in (B), (C), and (G) are representative of experiments repeated three times with similar results.

Techniques: Western Blot, Methylation, Knockdown, Quantitation Assay, Migration, Expressing, Mutagenesis, Construct, Activity Assay

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