Journal: STAR Protocols

Article Title: Protocol for assessing the role of hippocampal perineuronal nets in aversive memories

doi: 10.1016/j.xpro.2021.100931

Figure Lengend Snippet:

Article Snippet: Chondroitinase ABC , Amsbio , Cat#: E1028-10.

Techniques: Recombinant, Plasmid Preparation, Blocking Assay, Cell Culture, Fluorsave, Software, Microscopy

Journal: Biomolecules

Article Title: Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

doi: 10.3390/biom11060836

Figure Lengend Snippet: Elaiophylin disrupts the interaction between Cdc37 and the N-terminus of Hsp90, without binding to the ATP-pocket of Hsp90. ( A ) Chemical structure of elaiophylin. ( B ) Coimmunoprecipitation of Cdc37 with Hsp90 from MDA-MB-231 or MIA PaCa-2 cell lysates treated for 24 h with either 0.1% DMSO vehicle control, 1 μM elaiophylin, or 10 μM conglobatin A as indicated. Vehicle control treated cell lysate incubated with mouse IgG isotype control was used as a negative control; note the faint band in the Cdc37 probed line corresponds to the IgG molecular weight. ( C , D ) Elaiophylin was tested at increasing concentrations in a lysate-based split Renilla luciferase assay using NRL-Hsp90/ Cdc37-CRL ( C ) or NRL-N-Hsp90/ Cdc37-CRL ( D ). ( E ) Fluorescence polarization assay detecting competition of indicated compounds tested at 10 μM with fluorescently labelled geldanamycin for the ATP-pocket of Hsp90. ( F ) Computational docking of elaiophylin (cyan sticks) to the N-terminus of human Hsp90 (green; PDB ID 3T0Z). The interacting residues in Hsp90 are shown as gray sticks, ATP (magenta sticks) and magnesium (deep purple sphere) are indicated. The polar interactions between elaiophylin and the protein are shown as dashed lines with distances in Ångströms. The statistical significance levels are annotated in the plots as *** p < 0.001; **** p < 0.0001.

Article Snippet: Conglobatin A (cat. BIA-C1022), conglobatin B1 (cat. BIA-C1980), conglobatin C1 (cat. BIA-C2682), and elaiophylin (cat no BIA-E1028) were purchased from Bioaustralis.

Techniques: Binding Assay, Incubation, Negative Control, Molecular Weight, Luciferase, Fluorescence

Journal: Biomolecules

Article Title: Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

doi: 10.3390/biom11060836

Figure Lengend Snippet: Elaiophylin treatment alters biomarkers of a Hsp90/ Cdc37 inhibitor and selectively inhibits K-Ras nanoclustering. ( A , B ) Western blots for indicated antigens of KRAS mutant MDA-MB-231 ( A ) and MIA PaCa-2 ( B ) treated with 0.1% DMSO vehicle control, 2 μM 17-AAG, 10 μM conglobatin A, or indicated concentrations of elaiophylin. ( C , D ) Ras nanoclustering measured by FRET in HEK cells co-expressing mGFP- or mCherry-tagged K-RasG12V ( C ) or mGFP- or mCherry-tagged H-RasG12V ( D ). Cells were treated with 0.1% DMSO control, 2 μM conglobatin A, conglobatin B1, conglobatin C1, or 1 μM elaiophylin for 24 h. Bottom, Examples of FLIM-FRET images of HEK cells with indicated treatments. “Donor only” refers to cells transfected with mGFP-RasG12V only (no FRET control). ( E ) Chemical structures of analyzed conglobatins. The statistical significance levels are annotated in the plots as **** p < 0.0001.

Article Snippet: Conglobatin A (cat. BIA-C1022), conglobatin B1 (cat. BIA-C1980), conglobatin C1 (cat. BIA-C2682), and elaiophylin (cat no BIA-E1028) were purchased from Bioaustralis.

Techniques: Western Blot, Mutagenesis, Expressing, Transfection

Journal: Biomolecules

Article Title: Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

doi: 10.3390/biom11060836

Figure Lengend Snippet: Elaiophylin potently reduces MDA-MB-231 3D spheroid growth and microtumor formation in the CAM model. ( A , B ) Dose–response analysis of elaiophylin tested for 72 h on MDA-MB-231 and MIA PaCa-2 cells in 2D cell proliferation ( A ) and 3D spheroid formation assays ( B ); n = 2. Right of ( B ) shows example images of spheroid formation after treatment with indicated elaiophylin concentrations. ( C ) Microtumor formation of MDA-MB-231 cells on the chick CAM. Cells growing into microtumors were treated for 5 days with 0.1% DMSO vehicle control, 10 μM conglobatin A, or 5 μM elaiophylin. Right, shows example images of microtumors. The statistical significance levels are annotated in the plots as **** p < 0.0001.

Article Snippet: Conglobatin A (cat. BIA-C1022), conglobatin B1 (cat. BIA-C1980), conglobatin C1 (cat. BIA-C2682), and elaiophylin (cat no BIA-E1028) were purchased from Bioaustralis.

Techniques: