Journal: bioRxiv

Article Title: Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

doi: 10.1101/2022.12.04.519012

Figure Lengend Snippet: a. Annotated DDB1 and AMBRA1 domain schematics. AMBRA1 WD40 construct for cryo-EM was defined. BPA-BPC, β -propeller A-C; CTD, C-terminal helical domain; FL, full length; IDRs, intrinsically disordered regions. b. The coomassie blue stained SDS-PAGE analysis of purified AMBRA1 FL alone and AMBRA1 FL -DDB1-Cullin4A-RBX1 complex which were used for HDX-MS measurements. TSF, twin-strep-flag. c. Plot representing deuterium uptake by full length AMBRA1, with each data point representing the calculated central residue of an individual peptide. The Y-axis represents % deuteration for a given peptide, at each time point. HDX data statistics is given in . d. Isotopic envelopes for the selected peptides from AMBRA1, either alone or in E3 ligase complex following 3 s of incubation in D 2 O.

Article Snippet: The ubiquitination assays were performed in a 25 μl reaction volume with the following components: 100 nM UBE1 (Boston Biochem E-304), 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71).

Techniques: Construct, Cryo-EM Sample Prep, Staining, SDS Page, Purification, Residue, Incubation

Journal: bioRxiv

Article Title: Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

doi: 10.1101/2022.12.04.519012

Figure Lengend Snippet: Bimodal isotopic envelopes for the AMBRA1 N-terminal helix (peptide spanning residues 2-28), either alone or in E3 ligase complex. Deuteration time points are indicated. Close to open transition kinetics for AMBRA1 helix-loop-helix motif is calculated by fit of two gaussians to the high and low mass subpopulation. Relative amount of the open state is plotted against deuteration time. Structure of the AMBRA1-DDB1 complex interface showing the peptide spanning residues 2-28.

Article Snippet: The ubiquitination assays were performed in a 25 μl reaction volume with the following components: 100 nM UBE1 (Boston Biochem E-304), 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71).

Techniques:

Journal: bioRxiv

Article Title: Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

doi: 10.1101/2022.12.04.519012

Figure Lengend Snippet: a. The size exclusion profile (Superdex 200 Increase 10/300) and the coomassie blue stained SDS-PAGE analysis of the purified protein Cullin4A-DDB1-RBX1(E3) ligase which were used for in vitro ubiquitination assay. b. The size exclusion profile (Superose 6 Increase 10/300) and the coomassie blue stained SDS-PAGE analysis of the DDB1 which was used for MBP pull down assay. c. The coomassie blue stained SDS-PAGE analysis of the WT/truncated/mutated AMBRA1. d. The coomassie blue stained SDS-PAGE analysis of the Cyclin D1-CDK4 complex.

Article Snippet: The ubiquitination assays were performed in a 25 μl reaction volume with the following components: 100 nM UBE1 (Boston Biochem E-304), 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71).

Techniques: Staining, SDS Page, Purification, In Vitro, Ubiquitin Assay, Pull Down Assay

Journal: International journal of antimicrobial agents

Article Title: Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

doi: 10.1016/j.ijantimicag.2020.106099

Figure Lengend Snippet: A: Freshly fertilized eggs are transferred into a petri dish containing the test solution, viable eggs are selected microscopically, and 24 eggs containing viable embryos are transferred into a 24 well plates (1 egg per well). 20 wells contain 1 ml of BKI solution at a defined concentration (T), 4 wells serve as internal control (iC); B: 24 well-plate setup. T = BKI test concentration; nC = negative control (E3 medium/osmosis water); iC = internal control (E3 medium/osmosis water); sC = solvent control (DMSO); pC = positive control: dichloroaniline (4 g/L in water) (adapted from [31; 34])

Article Snippet: At 3h post fertilization (3hpf), zebrafish eggs were transferred into a petri dish (Sarstedt Inc, Nümbrecht, Germany) containing E3-medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 , and 0.33mM Mg SO 4 , buffered to pH7.5 using sodium-bicarbonate)/osmosis water) containing the tested drug concentration.

Techniques: Concentration Assay, Negative Control, Positive Control