Journal: eLife

Article Title: Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing associated with therapeutic response to splicing inhibitor

doi: 10.7554/eLife.90993

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-U2AF2 (rabbit polyclonal) , Novus Biologicals , NBP2-04140 , WB 1:1000.

Techniques: Recombinant, SYBR Green Assay, Real-time Polymerase Chain Reaction, Labeling, Transfection, Protease Inhibitor, Immunoprecipitation, RNA Immunoprecipitation, Plasmid Preparation, Expressing, Sequencing, shRNA, Knockdown, CRISPR, Software, Gene Expression, Alternative Splicing

Journal: Molecular cell

Article Title: The splicing factor CCAR1 regulates the Fanconi anemia/BRCA pathway

doi: 10.1016/j.molcel.2024.06.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-U2AF2 , Cell Signaling Technology , Cat# 70471; RRID: N/A.

Techniques: Recombinant, Transfection, Electroporation, Staining, Blocking Assay, cDNA Synthesis, Lysis, Protease Inhibitor, Immunoprecipitation, Sequencing, Modification, Electron Microscopy, Cell Viability Assay, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Knock-Out, Plasmid Preparation, Software, Functional Assay, Irradiation, Real-time Polymerase Chain Reaction, Mass Spectrometry

Journal: The Journal of Clinical Investigation

Article Title: AEP-cleaved DDX3X induces alternative RNA splicing events to mediate cancer cell adaptation in harsh microenvironments

doi: 10.1172/JCI173299

Figure Lengend Snippet: ( A ) Immunoblots of mCherry-tagged tDDX3X-C, lamin B and β-tubulin in the nuclear and cytoplasmic fractions of U87-MG-mCherry-tDDX3X-C cells. mCherry antibody (1:50) was used to precipitate mCherry-tDDX3X-C in the nuclear lysate of 2 cell samples; #1, sample 1; #2, sample 2; C, cytoplasmic; N, nuclear. ( B ) Venn diagram showing the intersection of 2 sets of proteins detected by IP-MS. There were 611 kinds of proteins in the intersection and 405 kinds of nuclear proteins are represented in green circle. ( C ) GO and KEGG term enrichment analyses of the DDX3X-interacting nuclear proteins identified from the IP-MS data. ( D ) String 10.5 program ( http://string-db.org ) analyses showing the interaction networks among 21 candidate proteins associated with mRNA splicing. ( E ) Expression correlation analysis of DDX3X and splicing factors based on the TCGA database. ( F ) HEK293T cells were cotransfected with eGFP-U2AF2/mCherry-tDDX3X-C. Scale bar: 40 μm. ( G ) Diagram of BIFC. ( H ) HEK293T cells were transfected with YN155-empty vector/YC156-tDDX3X-C, YN155-SRSF1/YC156-tDDX3X-C, or YN155-hnRNPA1/YC156-tDDX3X-C. Scale bar: 100 μm. ( I ) Co-IP assays of HEK293T cells transfected with the indicated plasmids. ( J ) U87-MG and MDA-MB-231 cells expressing tDDX3X-C (1 × 10 7 ) were harvested for Co-IP assays. ( K ) RT-qPCR assays of the indicated mRNAs in U87-MG and MDA-MB-231 cells. ( L ) PCR and AGE analyses for PRDM2 exon 2 in U87-MG and MDA-MB-231 in the following groups: NC, shSRSF1-3 and shhnRNPA1-1; quantification of percent spliced in (PSI). ( M ) PCR and AGE analyses for PRDM2 exon 2 and ARRB1 exon 13 in U87-MG and MDA-MB-231 grouped by NC, hnRNPA1 OE, and hnRNPA1 OE/shDDX3X-C-1. Data were plotted as the mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Dunnett’s multiple comparisons test ( K and L ) and Tukey’s multiple comparisons test ( M ). * P < 0.05, *** P < 0.001, **** P < 0.0001. Data shown are representative of 3 independent experiments.

Article Snippet: The primary antibodies are as follows: anti-DDX3X antibody (Proteintech, Cat 11115-1-AP, RRID: AB_10896499), anti-AEP antibody (R&D Systems, Cat AF2199, RRID: AB_416565), anti-Flag antibody (Sigma-Aldrich, Cat F1804, RRID: AB_262044), anti-Ki-67 antibody (Abcam, Cat ab16667, RRID: AB_302459), anti- mCherry antibody (Abcam, Cat ab167453, RRID: AB_2571870), anti-β-tubulin antibody (Abcam, Cat#ab21058, RRID: AB_727045), anti-Lamin B1 antibody (Proteintech, Cat 12987-1-AP, RRID: AB_2136290), anti-U2AF2 antibody (Santa Cruz Biotechnology, Cat sc-53942, RRID: AB_831787), anti-SRSF1 antibody (Santa Cruz Biotechnology, Cat sc-33652, RRID: AB_628248), anti-hnRNPA1 antibody (Santa Cruz Biotechnology, Cat sc-32301, RRID: AB_627729), and anti-β-actin antibody (Abcam, Cat ab8226, RRID: AB_306371).

Techniques: Western Blot, Protein-Protein interactions, Expressing, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: Cell systems

Article Title: BraInMap elucidates the macromolecular connectivity landscape of mammalian brain

doi: 10.1016/j.cels.2020.03.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used were (mouse) anti-TARDBP (Abnova; H00023435-M01; 1:500), (rabbit) anti-TDP-43 phosph-S409/410 (a gift from Leonard Petrucelli; Rb3655; 1:250), (rabbit) anti-DDX1 (ProteinTech; 11357-1-AP; 1:500), (rabbit) anti-DDX5 (Abcam; ab21696; 1:1000), (rabbit) anti-hnRNP-H (Bethyl; A300-511A; 1:500), (rabbit) anti-ILF3 (Bethyl; NF90/NF110, A303-121A), (rabbit) anti-U2AF2 (Novus; NBP2-04138), (rabbit) anti-Histone H3 (Abcam; ab18521), (chicken) anti-MAP2 (Aves; MAP2) and (chicken) anti-NeuN (EMD; ABN91; 1:500).

Techniques: Control, Virus, Mutagenesis, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Functional Assay, Gene Expression, RNA Binding Assay, Software, Sequencing, Mann-Whitney U-Test, Mass Spectrometry

Journal: Molecular cell

Article Title: Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1

doi: 10.1016/j.molcel.2019.07.017

Figure Lengend Snippet: Key Resources Table

Article Snippet: Primary antibodies were: anti-SF3B1 (Bethyl Laboratories, A300–996A, 1:1,000), anti-ACTIN (Sigma, A2066, 1:2,000), anti-HA rabbit polyclonal (Abm, G166, 1:1,000), anti-HA mouse monoclonal (Sigma, H3663, 1:1,000), anti-DYKDDDDK (GenScript, A00187, 1:1,000), anti-SUGP1 (Bethyl Laboratories, A304–675A-M, 1:1,000), anti-RBM6 (ABclonal, A10391, 1:1,000), anti-U2AF2 (Sigma, U4758, 1;10,000), and anti-GST (Invitrogen, A5800, 1:1,500).

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, CRISPR, Software

Journal: Cell

Article Title: Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription

doi: 10.1016/j.cell.2019.06.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-U2AF2 , Bethyl , A303-665A; RRID: AB_11204941.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Staining, Blocking Assay, Ligation, Purification, Gel Extraction, Imaging, Sequencing, Software