setd2-halotag human orf in pfn21a  (Promega)

Journal: bioRxiv

Article Title: The CNS Microenvironment Promotes Leukemia Cell Survival by Disrupting Tumor Suppression and Cell Cycle Regulation in Pediatric T-cell Acute Lymphoblastic Leukemia

doi: 10.1101/2023.09.01.555887

Figure Lengend Snippet: A) RNA-sequencing analysis of normalized FPKM values showing decreased expression of SETD2 in CNS3 diagnosis patients compared to CNS1 diagnosis samples. (Significance calculated by two-tailed, unpaired Student’s t-test. *p<0.05). B) RNA-sequencing correlation analysis of SETD2 and TP53 (left graph) showing a negative correlation (r=-0.694, **p=<0.01) and MDM2 (right graph) showing a positive correlation (r=0.628, *p=<0.05) in CNS relapsed T-ALL patients. (Correlation was calculated using Pearson correlation coefficients, two-tailed with 95% confidence interval). C) String diagram showing protein-protein interactions directly and indirectly experimentally linking SETD2 with TP53, MDM2, BCLX, BCL2, NANOG and OCT4. D) mRNA expression levels (fold change versus empty vector control) of MDM2, TP53, MYCC, OCT4, NANOG, BCL2 and BCLX 24 hours after SETD2 overexpression by transfection in MOLT16 cells. E) mRNA expression levels of SETD2 overexpression in T.-ALL cell lines MOLT16, Jurkat, CCL-119 negatively correlates with BCL2 (r=0.-995, p=0.066), BCLX (r=-0.969), MDM2 (r=-979) and TP53 (r=-0.993, p=0.075). (Correlation was calculated using Pearson correlation coefficients, two-tailed with 95% confidence interval).

Article Snippet: DH5α competent cells (Invitrogen) were transformed by heat shock in 42°C water bath using SETD2 Human Tagged ORF Clone (Origene, RG224760) or pCMV6□AC□GFP Mammalian Expression Vector (Origene, PS100010) followed by overnight culture on LB agar plates (Sigma-Aldrich) containing 100μg/ml ampicillin (Sigma-Aldrich) at 37°C.

Techniques: RNA Sequencing Assay, Expressing, Two Tailed Test, Plasmid Preparation, Control, Over Expression, Transfection

Journal: Nature Communications

Article Title: Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

doi: 10.1038/s41467-021-26799-3

Figure Lengend Snippet: a Multiple sequence alignment of previously identified SHI domain in SETD2. b Exemplary ITC titration data of hnRNP L RRM2 with SETD2 2167–2192 and its fitting curve are shown. K D dissociation constant, DP differential power, N binding stoichiometry. For the arithmetic mean of K D values of the three independent experiments and the thermodynamic parameters see Supplementary Table . All ITC binding curves are shown in Supplementary Table . c ITC fitting curves of hnRNP L RRM2 with SETD2 2167–2192 (black) and SETD2 2113–2140 (red) are shown. d Illustration showing the deletions in SETD2 SHI domain that were made to perform affinity purification. e Silver staining and f western blotting of affinity-purified complexes of SETD2C and its mutants. The experiment was repeated three times all yielding similar results. Source data are provided as a Source Data file.

Article Snippet: hnRNP L, hnRNP LL, and SETD2 human ORF were procured from Promega.

Techniques: Sequencing, Titration, Binding Assay, Affinity Purification, Silver Staining, Western Blot

Journal: Nature Communications

Article Title: Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

doi: 10.1038/s41467-021-26799-3

Figure Lengend Snippet: a Ribbon representations of hnRNP L RRM2 bound to the SETD2 2167–2192 peptide. hnRNP L is colored in purple and the bound SETD2 2167–2192 peptide is colored in orange. b The SETD2 2167–2192 peptide is represented as ribbons on the molecular face of hnRNP L RRM2. The sidechains of SETD2 Leu2188 and SETD2 Leu2189 are shown as sticks. Red and blue colors denote negative and positive surface charges, respectively. The electrostatic potential surfaces were generated with PyMol at the contouring value of the potential from −52.3 to 52.3 kTe − 1 . c – f Closeup views of the interactions between hnRNP L and the SETD2 2167–2192 peptide. (Left) The van der Waals surface views of hnRNP L-SETD2 2167–2192 . hnRNP L (purple) and SETD2 2167–2192 (orange) are shown as ribbons with selected sidechains as sticks. The van der Waals surface of the hnRNP L is depicted as a semitransparent skin. The SETD2 peptide is represented as a stick diagram (orange). (Right) Hydrogen bonds are shown as black dashed lines.

Article Snippet: hnRNP L, hnRNP LL, and SETD2 human ORF were procured from Promega.

Techniques: Generated

Journal: Nature Communications

Article Title: Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

doi: 10.1038/s41467-021-26799-3

Figure Lengend Snippet: a , d , h Cartoon illustrating the domain organization of hnRNP L, hnRNP LL, SETD2, and ySet2. b , c Heat maps showing the enrichment of proteins normalized to the bait (SETD2C) in MudPIT analysis. e , f , j Western blotting and silver staining of affinity-purified complexes of Halo-SETD2C or Halo-ySet2 and its mutants from 293 T extracts. The experiment was repeated at least three times all yielding similar results. Source data are provided as a Source Data file. g , i Table showing the dNSAFs of the listed proteins post mass spectrometry analysis of purified complexes obtained by affinity purification of Halo-SETD2 or ySet2 from 239 T extracts. AWS associated with SET, SET-Su(var)3-9 enhancer-of-zeste and trithorax, SRI–Set2–Rpb1 interaction, SHI– SETD2–hnRNP interaction dNSAF distributed normalized spectral abundance factor, NLS nuclear localization signal.

Article Snippet: hnRNP L, hnRNP LL, and SETD2 human ORF were procured from Promega.

Techniques: Western Blot, Silver Staining, Affinity Purification, Mass Spectrometry, Purification

Journal: Nature Communications

Article Title: Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

doi: 10.1038/s41467-021-26799-3

Figure Lengend Snippet: a Sequence alignment of hnRNP L RRM2 and hnRNP LL RRM2 proteins. The alignment was generated by ESPript3 with CLUSTALW . The secondary structures of hnRNP L RRM2, as determined by DSSP, are shown above the sequences. Red squares denote identical residues whereas black triangles highlight the key residues involved in the interaction with SETD2. b , e Halo purification was performed from extracts of 293 T cells co-expressing Halo-tagged SETD2C and mCherry-HA-hnRNP L/LL constructs. Input and eluted samples were resolved on gel followed by western blotting. The experiment was repeated at least 2 times all yielding similar results. Source data are provided as a Source Data file. c Halo purification was performed from extracts of 293 T cells co-expressing Halo-HA-tagged hnRNP LL and GFP-FLAG-SETD2C constructs. Input and eluted samples were resolved on gel followed by western blotting. The experiment was repeated at least two times all yielding similar results. Source data are provided as a Source Data file. d Schematic representation of hnRNP LL segments used in purification experiments. f ITC titration data of hnRNP LL with SETD2 2167–2192 and its fitting curve are shown. For the arithmetic mean of K D values of the three independent experiments and the thermodynamic parameters see Supplementary Table . All ITC binding curves are shown in Supplementary Table . g (Left) Structure comparison of hnRNP L_RRM2–SETD2 2167–2192 complex (orange) and hnRNP LL_RRM2–SETD2 2167–2192 complex (purple). (Right) Two complexes are shown as ribbons with selected sidechains as sticks. K D dissociation constant, DP differential power, N binding stoichiometry. RRM–RNA recognition motif, NLS nuclear localization signal.

Article Snippet: hnRNP L, hnRNP LL, and SETD2 human ORF were procured from Promega.

Techniques: Sequencing, Generated, Purification, Expressing, Construct, Western Blot, Titration, Binding Assay

Journal: Nature Communications

Article Title: Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

doi: 10.1038/s41467-021-26799-3

Figure Lengend Snippet: a ITC fitting curves of hnRNP L RRM2 WT and mutants with SETD2 2167–2192 peptide are shown. b , d , e Western blotting and silver staining of affinity-purified complexes of Halo-SETD2C WT and its mutants. In ( b ), mCherry-HA-hnRNP L proteins were used as prey. * denotes non-specific band. The experiment was repeated four times all yielding similar results. Source data are provided as a Source Data file. c , f ITC fitting curves of hnRNP L RRM2 with SETD2 peptides. For the arithmetic mean of K D values of the three independent experiments and the thermodynamic parameters see Supplementary Table . All ITC binding curves are shown in Supplementary Table .

Article Snippet: hnRNP L, hnRNP LL, and SETD2 human ORF were procured from Promega.

Techniques: Western Blot, Silver Staining, Affinity Purification, Binding Assay