Journal: Molecular Cell

Article Title: Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts

doi: 10.1016/j.molcel.2018.11.024

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Article Snippet: Gamma-32P-adenosinetriphosphate , Perkin Elmer , BLU502A100UC.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Protease Inhibitor, Magnetic Beads, Mutagenesis, Sequencing, Expressing, Software

Journal: Cell

Article Title: Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

doi: 10.1016/j.cell.2020.07.033

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Article Snippet: Isotope [α- 32 P] ATP , Perkin Elmer , Cat.# BLU003H250UC.

Techniques: Virus, Infection, Recombinant, Reverse Transcription, Software, Protease Inhibitor

Journal: Journal of Biological Chemistry

Article Title: Probing Conformational Rescue Induced by a Chemical Corrector of F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutant

doi: 10.1074/jbc.m111.239699

Figure Lengend Snippet: FIGURE 5. The effect of chemical correction and ablation of the R553XR555 sequence on conformational correction of F508del-CFTR probed using limited proteolysis. Limited proteolysis of control or chemically corrected F508del-CFTR, F508del-KXK-CFTR, or WT-CFTR with increasing levels of trypsin (0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 g/ml) was performed. Digests were analyzed by SDS-PAGE, and immunoreactive bands were detected using the Odyssey infrared imaging system from LI-COR Biosciences. The colored signal was converted to grayscale for the preparation of this figure. A, samples were immuno- blotted with anti-CFTR monoclonal antibody, L12B4 (NBD1-specific). Subsequent analyses focused on the larger NBD1 fragments (34–36 kDa) and the smaller NBD1 fragments (lower than 28 kDa). Both sets of fragments were indicated using brackets on the right-hand side of each image. B, samples were immuno- blotted with anti-CFTR monoclonal antibody, M3A7 (NBD2-specific). NBD2 fragments of 28 kDa were analyzed subsequently (indicated using brackets on the right-hand side of each image).

Article Snippet: L12B4 anti-CFTR antibody (1:1000) was used for immunodetection conjugated to IRDye800 (1:10 000) (Rockland Immunochemical Inc).

Techniques: Sequencing, Control, SDS Page, Imaging

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: (A) U2OS reporter cell line transfected with indicated siRNA SMARTpools was treated with Shield-1 and 4-OHT for 5 h to induce DSBs by mCherry-Lac1-Fok1. Colocalization of BRCA1 or 53BP1 with Fok1 was imaged by IF, scale, 200 M. Representative experiment from n=3 each for 53BP1 and BRCA1. Quantitation of 15–20 fields from each sample is depicted, ****, p<0.0001, n.d.- not detected (lower left panel); A.U.- Arbitrary units. Re-drawn schematic of the reporter assay, previously published (Tang et al., 2013) is depicted (lower right panel) (B) BRCA1 colocalization with Fok1 was examined in 53BP1-deficient U2OS reporter cells. (See also Figure S2B.) n.s.- not significant; A.U.- Arbitrary units. (C) AcH4 ChIP using different primer sets for endogenous loci was performed in a U2OS-TRF1-Fok1 reporter cell line that induces DSBs within telomeres (Tang et al., 2013). DSBs were induced by treatment with 4-OHT for 5 h; mean +/− SEM for 5 h, *, p<0.05, **, p<0.01, n.s.- not significant, n.d.- not detected. See Figure S3 for primer locations. (D) HR assay performed in the DR-GFP U2OS reporter line (Pierce et al., 2001). Cells were transfected with individual siRNAs for 24 hours (siACLY A or siACLY B), followed by transfection of the endonuclease Sce-1 for an additional 48 h. GFP positivity, indicating HR efficiency, was analyzed by flow cytometer, mean +/− SEM, **, p<0.01; ***, p<0.001. (E) To assay HR, cells were transduced with EV or ACLY-expressing lentivirus. Cells were transfected with siRNA SMARTpools, followed by Sce-1 as in (D). Data is representative of 2 independent experiments. Mean +/− SEM is graphed, ***, p<0.001. (F) ACLY was silenced using two independent shRNAs in HeLa cells expressing TRF2ΔB/ΔM. NHEJ was assessed by scoring end-to-end chromosomal fusions. Mean +/− SEM **, p<0.01, ***,p<0.001. See also Figure S2 and Figure S3.

Article Snippet: HR assay To examine HR, DR-GFP reporter cells were plated at 30,000–50,000 cells/well in a 12 well plate and reverse transfected with either control hairpin or individual siRNAs targeting ACLY (Origene) with RNAiMax (Thermo Fischer Scientific) using standard protocol.

Techniques: Transfection, Quantitation Assay, Reporter Assay, Flow Cytometry, Transduction, Expressing