Journal: bioRxiv

Article Title: Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

doi: 10.1101/2020.07.31.231274

Figure Lengend Snippet: a. Diagram showing the domain architecture of SARS-CoV-2 nsp13. b. Size exclusion chromatography (Superdex 200 Increase) of purified nsp13. c. SDS-PAGE gel (Coomassie stain) showing nsp13 purity. d. Differential scanning fluorimetry (DSF) of SARS-CoV-2 nsp13 in the absence and presence of ATP analogs (1 mM). Melting temperatures are: APO- 40.5 °C, ATPγS- 45.5 °C, ADP:AlF 3 - 52.5 °C, and ADP- 41.0 °C for n=2 independent measurements from two separate protein preparations. e. Fluorescence anisotropy measurements for fluorescence DNA and RNA partial duplexes (10 nM) with and without nsp13 (3.4 μM) added. Data shown as mean standard ± deviation (SD) for n=2 independent measurements from two separate protein preparations.

Article Snippet: These experiments were carried out on a C1000 Touch Thermal cycler CFX-96 instrument (GE Healthcare). recombinant constructs were diluted to a final concentration of 4 μM in DSF buffer (25 mM HEPES-KOH pH 7.5, 25 mM KCl, 5 mM MgCl 2 , 1 mM TCEP, 0.005% triton-X) and assayed in a 96-well plate (Hard-shell HSP9665 Bio-Rad).

Techniques: Size-exclusion Chromatography, Purification, SDS Page, Staining, Fluorescence, Standard Deviation

Journal: Infection and Immunity

Article Title: GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii

doi: 10.1128/IAI.00180-20

Figure Lengend Snippet: Differential scanning fluorimetry analysis of GigC. Differential scanning fluorimetry (DSF) assays were performed on recombinant GigC alone (A and B) and in the presence of the indicated concentrations of l-cysteine (l-Cys) (C and D). DSF data are plotted as the first derivative of the raw fluorescence value over the indicated temperature range. The DSF experiment was repeated three times, and results from a representative experiment are shown.

Article Snippet: Briefly, 20-μl reaction mixtures were prepared in clear 96-well microtiter plates (Applied Biosystems) in DSF buffer (9.25 mM HEPES, 140 mM NaCl, 7.5× SYPRO orange [Sigma-Aldrich] [pH 7.0]).

Techniques: Recombinant, Fluorescence

Journal: bioRxiv

Article Title: Structural basis of NSD2 degradation via targeted recruitment of SCF-FBXO22

doi: 10.1101/2025.08.29.673087

Figure Lengend Snippet: (a) UNC10088 C -induced conformational change of the loop adjacent to C326. Structural overlay of the SCF FBXO22 -UNC10088 C -NSD2 PWWP1 model (dark gray) and the SCF FBXO22 -BACH1 BTB complex (light gray, PDB: 8UA3) , revealing the displacement of FBXO22 residues Y390. (b) Chemical structures of UNC8732 analogs that contain an aromatic aldehyde in place of the aliphatic amine of UNC8732. (c) Benzaldehyde derivatives UNC10415667 and UNC10415668 promote FBXO22-dependent ubiquitination of *NSD2 PWWP1 when added at 0.5 µM, based on fluorescent scanning of an SDS-PAGE gel. (d) Treatment of U2OS NSD2-HiBit cells with UNC10415667 (1 µM) for 6 hours resulted in a potent reduction of NSD2 levels by about 75%. A more modest effect was observed upon treatment with UNC10415668, and all other compounds had no significant effect. n ≥ 3 independent experiments. Error bars: standard error of the mean. (e) Treatment of U2OS NSD2 HiBit cells with UNC10415667 in a dose response fashion revealed a DC 50 value of 0.46 µM, which is about 2-fold more potent than UNC8732 and UNC10088. n = 3 independent experiments. Error bars: standard error of the mean. (f) UNC10415667 uses C326 to recruit NSD2 to the FBXO22 FIST domain as monitored by size-exclusion chromatography. Chromatograms of different elution profiles between mixtures of WT FBXO22 FIST or a C326A mutant with NSD2 and UNC10415667 (top). Representative Coomassie-stained SDS-PAGE gels of collected fractions. (g) Differential scanning fluorimetry (DSF) was used to monitor the stability of FBXO22 FIST wild type and variants in the presence of UNC10088 or UNC10415667 compared to DMSO, revealing that both compounds stabilize WT FBXO22 FIST and the C326 single cysteine variant (C326 only) as evidenced by an increase in melting temperature (ΔT m ), but not the C326A mutant. n = 3 independent experiments. Error bars: standard error of the mean.

Article Snippet: 9.5 μL of the master mix containing FBXO22 FIST WT or variants, SYPRO Orange Dye (Invitrogen), and DSF buffer (20 mM Tris 7.5, 150 mM NaCl, and 1 mM TCEP) were added to a 384-well qPCR plate (Genesee Scientific).

Techniques: Ubiquitin Proteomics, SDS Page, Size-exclusion Chromatography, Mutagenesis, Staining, Variant Assay