![]() ( E) Top: Autoradiogram of an SDS-PAGE gel in which the 5’-ends of IRE1-crosslinked RNAs were radiolabeled with polynucleotide kinase after immunoprecipitation of epitope-tagged IRE1. ER stress was induced by exposing the cells to tunicamycin. ( D) Semi-quantitative RT-PCR showing the extent of XBP1 splicing in the HEK293Trex cell line where we force-express IRE1 in the presence or absence of chemically-induced ER stress. ![]() ( C) Immunoblot showing the relative enrichment for IRE1 in native and denaturing IP conditions. ( A) Schematic of modified PAR-CLIP protocol. ( B) Immunoblots showing the enrichment for IRE1 in subcellular fractions prepared by differential detergent extraction as performed in our PAR-CLIP experiments. ‘Unknown region’ refers to transcripts associated with coding loci but that do not have a single annotated coding sequence ( i.e., alternative splicing or alternative transcription initiation sites). The diagonal dashed line indicates a slope of 1. ( F) Breakdown of mRNA regions associated with IRE1 in PAR-CLIP experiments. The histograms above and to the side of the scatter plot illustrate the frequency of transcripts that traverse the secretory pathway. ( E) Scatter plot showing the abundance of PAR-CLIP recovered transcripts in the presence or absence of chemically induced ER stress (copies per million reads: geometric mean of copy number per transcript in biological duplicates). Cardinals indicate the total or non-coding (in parenthesis) number of transcripts in each group. ![]() ( D) Venn diagrams showing the numbers of crosslinked transcripts recovered by PAR-CLIP in the presence or absence of chemically induced ER stress in biological duplicates. ( C) Breakdown of RNA classes associated with IRE1 identified by PAR-CLIP in biological duplicates. ( B) Mutation plots showing T→C transitions are the most common mutations recovered by PAR-CLIP in biological duplicates.
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