Journal: Cell reports

Article Title: SART3 reads methylarginine-marked glycine- and arginine-rich motifs

doi: 10.1016/j.celrep.2024.114459

Figure Lengend Snippet: (A) Schematic representation of the GFP-Trap-based IP-mass spectrometry (MS) approach. (B) List of GFP-RMET interacting proteins identified by IP-MS. HEK293T cells were transfected with GFP-RMET, and half the cells were treated with AdOx for 3 days and subjected to IP-MS analysis. The number of peptides identified for each protein is listed. (C) Far-western analysis of the interaction between a GST-SART3(HAT) and RMET. HEK293T cells were transfected with GFP-EV (empty vector) or the GFP-RMET fusion. After a 3-day treatment with AdOx, total lysates were probed with GST-SART3(HAT) and detected with anti-GST antibody. (D) GST-SART3(HAT) pull-downs using biotinylated fibrillarin GAR peptides. GST-TDRD3(Tudor) and GST-SMN(Tudor) are controls. A mild buffer (150 mM NaCl) was used. (E) GST-SART3(HAT) pull-downs using biotinylated coilin GAR peptides and using mild buffer (300 mM NaCl) and RIPA. Experiments (C–E) were independently repeated three times, with similar results.

Article Snippet: Fibrillarin peptide Rme2s (SDMA) GG[Rme2s]G[Rme2s]GGGF[Rme2s] G[Rme2s]G[Rme2s]GGGG-Biotin , CPC Scientific , N/A.

Techniques: Mass Spectrometry, Transfection, Western Blot, Plasmid Preparation

Journal: Cell reports

Article Title: SART3 reads methylarginine-marked glycine- and arginine-rich motifs

doi: 10.1016/j.celrep.2024.114459

Figure Lengend Snippet: (A) Western blot analysis of SART3 expression in CRISPR-mediated SART3-knockout (KO) HeLa cells and the stable restoration with GFP fusions to WT and mutant (Y112A and W377A) SART3. (B) IP/western blot analysis of the SART3 interaction with coilin and fibrillarin. Restored HeLa SART3-KO cells in (A) were used. (C) IP/western blot validation of the interaction between SART3 and a subset of spliceosome proteins identified by IP-MS. Restored HeLa SART3-KO cells in (A) were used. (D) Sashimi plots depicting exon skipping in the representative genes TMEM255B and KIAA0895 in HeLa SART3-KO cells restored with WT (red) and mutant (purple) SART3. False discovery rate < 0.05. (E) Semiquantitative RT-PCR supporting the TMEM255B and KIAA0895 exon skipping analysis using HeLa control cells (endogenous SART3), SART3-KO control cells (no SART3), and HeLa sgSART3-KO cells restored with WT GFP-SART3 or the Y112A and W377A mutants (restored), as shown in (A). Three biological repeats were performed ( n = 3). The PCR products were quantified by densitometric analysis, and the exon skipping ratio is shown in a percentage expressed as the mean ± SEM. Unpaired t test was used to calculate p values: ** p < 0.01 and *** p < 0.001.

Article Snippet: Fibrillarin peptide Rme2s (SDMA) GG[Rme2s]G[Rme2s]GGGF[Rme2s] G[Rme2s]G[Rme2s]GGGG-Biotin , CPC Scientific , N/A.

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cell reports

Article Title: SART3 reads methylarginine-marked glycine- and arginine-rich motifs

doi: 10.1016/j.celrep.2024.114459

Figure Lengend Snippet:

Article Snippet: Fibrillarin peptide Rme2s (SDMA) GG[Rme2s]G[Rme2s]GGGF[Rme2s] G[Rme2s]G[Rme2s]GGGG-Biotin , CPC Scientific , N/A.

Techniques: Virus, Recombinant, Protease Inhibitor, Membrane, Mutagenesis, SYBR Green Assay, Knock-Out, Plasmid Preparation, Software