ABSTRACT: MLF2-APEX2 fusion-based proximity labeling of nuclear envelope bleb composition. APEX reactions were conducted in WT and TorsinKO HeLa cells.
Project description:Datasets generated using WT or TorsinKO HeLa cells expressing APEX2-MLF2-HA or d133 ORF10 from the KSHV. Data were used to generate Table S1.
Project description:Proximity labeling approach to identify protein inside nuclear envelope blebs arising in Torsin-deficient HeLa cells. WT and TorsinKO cells were engineered to express MLF2-APEX2 fusion. APEX reaction was carried out via 1 mM H2O2 for 1 minute in the presence of biotin phenol. Biotinylated protein were captured via streptavidin beads. Each APEX reaction was accompanied by an untreated (no H2O2) control.
Project description:The truncated d133-ORF10-FLAG from KSHV was expressed and immunoprecipitated from WT and TorsinKO HeLa cells. The datasets were compared to an untransfected control. As d133-ORF10 localizes to blebs in TorsinKO cells, this approach allows probing of bleb protein contents.
Project description:HSPA1A was immunoprecipitated from WT or TorsinKO HeLa cells. A band around 27 kDa was uniquely found to co-immunoprecipitate with HSPA1A in TorsinKO cells. To identify this band, it was extracted and subjected to mass spectrometry.
Project description:HSPA1A was immunoprecipitated from WT or TorsinKO HeLa cells. A band around 27 kDa was uniquely found to co-immunoprecipitate with HSPA1A in TorsinKO cells. To identify this band, it was extracted and subjected to mass spectrometry.
Project description:MLF2 was purified from mammalian Expi293 cells as a fusion with MBP. As MLF2 associates tightly with HSP70/HSPA1A, this resulted in a stoichiometric co-purification with HSP70 members. To identify which ones, we subjected the whole purification or a gel-extracted band around 70 kDa to mass spectrometry.
Project description:We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
Project description:MLF2 was purified from mammalian Expi293 cells as a fusion with MBP. As MLF2 associates tightly with HSP70/HSPA1A, this resulted in a stoichiometric co-purification with HSP70 members. To identify which ones, we subjected the whole purification or a gel-extracted band around 70 kDa to mass spectrometry.