human setd2 catalytic domain  (Addgene inc)

Journal: bioRxiv

Article Title: Extrinsic MMPs drive epithelial shape change via basal ECM disassembly in the Drosophila wing disc

doi: 10.64898/2026.01.21.700823

Figure Lengend Snippet: (A–B’’) Wing discs carrying mmp1-GFP transcriptional reporter (green) and expressing RedStinger under the control of 1151-Gal4 (magenta) or btl-Gal4 (cyan) were dissected at 1–1.5 h after puparium formation (APF) and stained with anti-GFP antibody and DAPI (white). Orange arrowheads indicate Mmp1-positive myoblasts or tracheal cells. An asterisk indicates a Mmp1-positive tracheal cell shown in (A). Scale bar, 50 μm. (C–C’’) A wing disc carrying the Mmp2::GFP knock-in reporter (green) and expressing RedStinger under the control of 1151-Gal4 (magenta) at 1–1.5 h APF, stained with anti-GFP antibody and DAPI (white). Orange arrowheads indicate Mmp2-positive myoblasts. Scale bar, 50 μm. (D–D’’) A wing disc carrying the Mmp2::GFP knock-in reporter (green) at 1–1.5 h APF, stained with anti-GFP and anti-Gasp (cyan) antibodies, together with DAPI (white). Orange dotted lines outline the tracheal tube. Orange arrowheads indicate Mmp1-positive tracheal cells. Scale bar, 50 μm. (E–H’) Wing discs carrying viking::GFP (pseudocolor) were dissected, treated with ecdysone, and stained with anti-GFP antibody and DAPI (white). Images are shown for control (E) and for discs expressing Timp under the control of 1151-Gal4 (F), btl-Gal4 (G), or both 1151-Gal4 and btl-Gal4 (H). Yellow dotted lines outline the notum regions. Scale bar, 50 μm. (I) Dot plots showing the percentage of Collagen IV-disassembled area in the notum, where Collagen IV signals could be more reliably quantified than in the hinge region, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (E) (n = 23), (F) (n = 15), (G) (n = 21), and (H) (n = 14). *p<0.05, ****p<0.0001; pairwise comparisons using the Wilcoxon rank-sum test with BH correction. (J–M) Time-lapse confocal imaging of the ecdysone-treated wing discs carrying ubi-RFP (magenta). Images are shown for control (J) and for discs expressing Timp under the control of 1151-Gal4 (K), btl-Gal4 (L), or both 1151-Gal4 and btl-Gal4 (M). Orange arrowheads indicate the same anatomical position before and after the curvature change, marking the region that normally undergoes the concave-to-convex transition. Time is shown as h:min from the onset of ecdysone treatment. Scale bar, 100 μm. (N) Quantification of the final wing disc shape, shown as the percentage of discs that remained concave or transitioned to a convex shape, corresponding to the representative images shown in (J) (n = 44), (K) (n = 32), (L) (n = 35), and (M) (n = 20). *p<0.05, **p<0.01, ****p<0.0001; Fisher’s exact test with Holm correction.

Article Snippet: Primary antibodies used are as follows; rat anti-GFP antibody (Nacalai Tesque, #04404-26, 1:1000), rabbit anti-GFP antibody (Invitrogen, #A6455, 1:500), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) antibody (CST, #3671, 1:100), rabbit anti-Zfh1 antibody (R. Lehmann, 1:1000), rabbit anti-DsRed antibody (Clontech, # 632496, 1:500), mouse anti-Gasp antibody (DSHB, #2A12, 1:100), anti-Mmp1 antibody (DSHB, 1:10 from 1:1:1 cocktail of 3A6B4, 3B8D12 and 5H7B11) (PMID: 25224221), and Rabbit anti-Collagen IV antibody (1:100) (PMID: 40570847).

Techniques: Expressing, Control, Staining, Knock-In, Imaging

Journal: bioRxiv

Article Title: Extrinsic MMPs drive epithelial shape change via basal ECM disassembly in the Drosophila wing disc

doi: 10.64898/2026.01.21.700823

Figure Lengend Snippet: (A–D’’) Wing discs were dissected, treated with ecdysone, and stained with anti-Mmp1 (green) and anti-Zfh1 (magenta) antibodies. Images are shown for discs expressing empty-RNAi (VDRC60100) (A–B’) or EcR-RNAi (NIG1765R-2) (C–D’), together with Dicer2 (Dcr2), under the control of 1151-Gal4 and btl-Gal4 . (B–B’) and (D–D’) show magnified views of the regions outlined by cyan dotted lines in (A–A’) and (C–C’), respectively. Larvae carrying Tub-Gal80 ts were maintained under the temperature conditions illustrated in Fig. S5A. Orange arrowheads indicate the anti-Mmp1-positive puncta surrounding Zfh1-positive myoblast nuclei. Scale bar, 10 μm. (E) Dot plots showing the mean number of anti-Mmp1-positive puncta per myoblast, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (A–B) (n = 10) and (C–D) (n = 9). ****p<0.0001; Wilcoxon rank-sum test. (F–G’) Wing discs carrying Mmp2::GFP knock-in reporter (pseudocolor) were dissected, treated with ecdysone, and stained with anti-GFP and anti-Zfh1 (magenta) antibodies. Images are shown for discs expressing yellow-RNAi (NIG3757R-1) (F–F’) or EcR-RNAi (NIG1765R-2) (G–G’), together with Dcr2, under the control of 1151-Gal4 and btl-Gal4 . Larvae bearing Tub-Gal80 ts were maintained under the temperature conditions indicated in Fig. S5A. Scale bar, 50 μm. (H) Dot plots showing the mean intensity of Mmp2::GFP knock-in reporter in myoblasts, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (F) (n = 8) and (G) (n = 11). *p<0.05; Wilcoxon rank-sum test. (I–J’) Wing discs were dissected, treated with ecdysone, and stained with anti-Collagen IV antibody (pseudocolor) and DAPI (white). Images are shown for discs expressing empty-RNAi (VDRC60100) (I) or EcR-RNAi (NIG1765R-2) (J), together with Dcr2, under the control of 1151-Gal4 and btl-Gal4 . Larvae carrying Tub-Gal80 ts were maintained under the temperature conditions illustrated in Fig. S5A. Scale bar, 50 μm. (K) Dot plots showing the percentage of the Collagen IV-disassembled area in the notum, with horizontal bars indicating the mean ± SD, corresponding to the representative images shown in (I) (n = 17) and (J) (n = 17). **p<0.01; Wilcoxon rank-sum test. (L–N’) Wing discs were dissected and treated with ecdysone for 24 h. Images are shown for discs expressing empty-RNAi (VDRC60100) (L), EcR-RNAi (NIG1765R-2) (M), or EcR-RNAi (BDSC9327) (N), together with Dcr2, under the control of 1151-Gal4 and btl-Gal4 . Larvae carrying Tub-Gal80 ts were maintained under the temperature conditions illustrated in Fig. S5A. Schematic diagrams (L’, M’ and N’) illustrate the concave and convex shapes corresponding to the representative images shown in (L), (M), and (N). (O) Quantification of the final wing disc shape, shown as the percentage of discs that remained concave or transitioned to a convex shape, corresponding to the representative images shown in (L) (n = 79), (M) (n = 48), and (N) (n =53). *p<0.05; Fisher’s exact test with Holm correction.

Article Snippet: Primary antibodies used are as follows; rat anti-GFP antibody (Nacalai Tesque, #04404-26, 1:1000), rabbit anti-GFP antibody (Invitrogen, #A6455, 1:500), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) antibody (CST, #3671, 1:100), rabbit anti-Zfh1 antibody (R. Lehmann, 1:1000), rabbit anti-DsRed antibody (Clontech, # 632496, 1:500), mouse anti-Gasp antibody (DSHB, #2A12, 1:100), anti-Mmp1 antibody (DSHB, 1:10 from 1:1:1 cocktail of 3A6B4, 3B8D12 and 5H7B11) (PMID: 25224221), and Rabbit anti-Collagen IV antibody (1:100) (PMID: 40570847).

Techniques: Staining, Expressing, Control, Knock-In

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: The Journal of Biological Chemistry

Article Title: CAPN15 is a non-proteasomal, ubiquitin-directed calpain protease that regulates cell adhesion by cleaving E-cadherin

doi: 10.1016/j.jbc.2025.111034

Figure Lengend Snippet: CAPN15 recognizes the ubiquitinated cadherin-catenin complex via the NZF domains. A , differential interference contrast images of HCT116, KO, WTtg, CStg, and NEtg cells. Scale bar, 20 μm. B , the mean log 2 fold change of the protein abundance (CStg versus NEtg immunoprecipitates) with -log 10 p value of the interactome was shown on the x- and y-axes, respectively. Significantly increased proteins were defined as those with the value of the log 2 fold change >1 with p value < 0.05 (plots in a colored area). It should be noted that there was no change in abundance of CAPN15 in the CStg and NEtg immunoprecipitates. C , the heatmap shows the relative abundance of cadherins and catenins in the KO, WTtg, CStg, and NEtg immunoprecipitates. D , KO, WTtg, CStg, and NEtg cells were treated with dimethyl sulfoxide (DMSO; vehicle) or 1 μM MLN7243. Cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody, followed by western blotting. Asterisks indicate nonspecific bands. Vertical lines indicate the ubiquitinated forms. E , immunoprecipitated fraction from DMSO (vehicle)-treated CStg cells was incubated with or without ubiquitin-specific protease 2 (USP2cc), followed by western blotting. The asterisk indicates the non-specific band. Vertical lines indicate the ubiquitinated forms.

Article Snippet: Anti-FLAG immunoprecipitate prepared from CStg cells was incubated with 1 μM USP2 catalytic domain (USP2cc; R&D Systems) for 1 h at 37 °C.

Techniques: Quantitative Proteomics, Immunoprecipitation, Western Blot, Incubation, Ubiquitin Proteomics