paper n a pcdna3 flag sf3b1 addgene addgene  (Addgene inc)

Journal: Genes & Development

Article Title: Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription

doi: 10.1101/gad.341545.120

Figure Lengend Snippet: CDK7 inhibition with SY-351 causes widespread defects in splicing. ( A ) Differentially expressed mRNAs following SY351 treatment (DESeq2). Replicate RNA-seq data sets were generated from rRNA-depleted total RNA from HL60 cells treated with DMSO (CTRL) or SY-351 (50 nM in DMSO) for 5 h. ( B ) GSEA analysis. ( C ) SY-351 favors exon inclusion over exon skipping, splicing over retention of introns, and alters 5′ and 3′ splice site selection equally, without preference for upstream or downstream sites. Alternative splicing events significantly affected by SY-351 were identified in replicate RNA-seq data sets using MAJIQ . ( D ) IGV genome browser Sashimi plot of a segment of the LTV1 gene. Normalized read numbers for DMSO control and SY-351 treated samples are shown on the Y -axis. Splice junction read numbers for sense strand transcripts are shown in blue and red. Note reduced exon inclusion (green arrow) and retention of both flanking introns (black arrows) in SY-351. ( E ) Immunofluorescence microscopy of endogenous SF3B1 (green) and Hoechst (blue) in DMSO and SY-351 conditions. ( F ) Quantitation of SF3B1 puncta per nucleus in DMSO and SY-351 conditions, P = 0.001 ( n = 3 biological replicates).

Article Snippet: The expression plasmid was purchased from Addgene (pCDNA3.1-FLAG-SF3B1-WT 82576).

Techniques: Inhibition, RNA Sequencing Assay, Generated, Selection, Alternative Splicing, Control, Immunofluorescence, Microscopy, Quantitation Assay

Journal: Genes & Development

Article Title: Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription

doi: 10.1101/gad.341545.120

Figure Lengend Snippet: CDK7 kinase activity is negatively regulated by TFIIH. ( A ) SYPRO-stained gel of human TFIIH. ( B ) TFIIH activates CDK7 kinase toward the CTD, but reduces or prevents CDK7 phosphorylation of DSIF, SF3B1, U2AF2, and TFIIF. Within the CAK, CDK7 is activated toward these substrates. ( C ) SYPRO-stained gel of human CAK complex (MAT1 is GST-tagged). ( D ) Quantitation of CDK7 kinase results across biological replicates ( n = 3) with autorad signal normalized to CAK phosphorylated DSIF; P -values as shown.

Article Snippet: The expression plasmid was purchased from Addgene (pCDNA3.1-FLAG-SF3B1-WT 82576).

Techniques: Activity Assay, Staining, Quantitation Assay