Journal: Cells

Article Title: WWOX Induction Promotes Bcl-X L and Mcl-1 Degradation Through a Lysosomal Pathway upon Stress Responses

doi: 10.3390/cells15030270

Figure Lengend Snippet: WWOX promotes Bcl-X L and Mcl-1 protein downregulation through a lysosomal degradation pathway. ( A ) Wwox +/+ MEFs were treated with serum-free medium for 36 h with or without the presence of a proteasome inhibitor, MG132 (1 μM), during the last 18 h of serum starvation. ( B ) Wwox +/+ MEFs were treated with serum-free medium for 32 h with or without the presence of NH 4 Cl (25 mM) or a lysosome inhibitor, chloroquine (CQ; 100 μM), during the last 4 and 8 h of serum starvation. ( C ) Wwox +/+ MEFs were treated with serum-free medium for 32 h with or without the presence of a lysosomal enzyme inhibitor E64d (30 μM) or pepstatin A (15 μM) during the last 4 and 8 h of serum starvation. Bcl-X L , Mcl-1, and WWOX protein expression was examined by Western blotting. β-actin was used as a loading control. Inhibition of Itch protein degradation in cycloheximide (CHX)-treated cells was used as a MG132 reagent control. The representative results from four repeated experiments are shown. Densitometric analysis for quantification of protein levels is shown in .

Article Snippet: Pepstatin A was obtained from Tocris (Bristol, UK).

Techniques: Expressing, Western Blot, Control, Inhibition

Journal: Proteins: Structure, Function, and Genetics

Article Title: The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease

doi: 10.1002/(SICI)1097-0134(19990801)36:2%3C147::AID-PROT2%3E3.0.CO;2-3

Figure Lengend Snippet: Fig. 1. The dependence of the structural stability of the HIV-1 protease on the concentration of the inhibitor acetyl pepstatin. Shown in the figure is the heat capacity function measured at a protease concentration of 0.3 mg/mL in 10 mM glycine pH 3.6. The transition temperature increases as a function of acetyl pepstatin concentration. In these experiments, acetyl pepstatin concentrations were 0, 15, 25, 50, 100, 150, and 200µM respectively. In each case, the solid line corresponds to the best fit obtained with the two-state folding/dimerization model.

Article Snippet: HIV-1 protease (at 0.3 mg/mL) and different concentrations of acetyl pepstatin (Peptides International, Louisville, KY) was examined for thermal denaturation at a scanning rate of 1°C/min, from 25°C to 85°C.

Techniques: Concentration Assay

Journal: Proteins: Structure, Function, and Genetics

Article Title: The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease

doi: 10.1002/(SICI)1097-0134(19990801)36:2%3C147::AID-PROT2%3E3.0.CO;2-3

Figure Lengend Snippet: Fig. 2. Calorimetric titration of HIV-1 protease with the peptide acetyl pepstatin. A shows the heat effects associated with the injection of acetyl pepstatin (10µL per injection of a 280 µM solution) into the calorimetric cell (1.4 mL) containing HIV-1 protease at a concentration of 20µM. The experiment was performed at 25°C. B shows the binding isotherm corresponding to the data in A and the best fitted curve.

Article Snippet: HIV-1 protease (at 0.3 mg/mL) and different concentrations of acetyl pepstatin (Peptides International, Louisville, KY) was examined for thermal denaturation at a scanning rate of 1°C/min, from 25°C to 85°C.

Techniques: Titration, Injection, Concentration Assay, Binding Assay

Journal: Proteins: Structure, Function, and Genetics

Article Title: The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease

doi: 10.1002/(SICI)1097-0134(19990801)36:2%3C147::AID-PROT2%3E3.0.CO;2-3

Figure Lengend Snippet: Fig. 3. Temperature dependence of the thermodynamic parameters for acetyl pepstatin binding to the HIV-1 protease. Note that at tempera- tures lower than 40°C, the Gibbs energy of binding is not favored enthalpically and that at higher temperatures the situation is reversed. At physiological temperatures and below the binding affinity increases with temperature.

Article Snippet: HIV-1 protease (at 0.3 mg/mL) and different concentrations of acetyl pepstatin (Peptides International, Louisville, KY) was examined for thermal denaturation at a scanning rate of 1°C/min, from 25°C to 85°C.

Techniques: Binding Assay

Journal: Proteins: Structure, Function, and Genetics

Article Title: The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease

doi: 10.1002/(SICI)1097-0134(19990801)36:2%3C147::AID-PROT2%3E3.0.CO;2-3

Figure Lengend Snippet: Fig. 4. The dependence of the denaturation temperature of the protease molecule on acetyl pepstatin concentration. The solid lines were calculated with the master equation for the Gibbs energy for the structural stability of the HIV-1 protease (equation 4) and the linkage equation (equations 5, 6) with the thermodynamic values indicated in the text.

Article Snippet: HIV-1 protease (at 0.3 mg/mL) and different concentrations of acetyl pepstatin (Peptides International, Louisville, KY) was examined for thermal denaturation at a scanning rate of 1°C/min, from 25°C to 85°C.

Techniques: Concentration Assay

Journal: Proteins: Structure, Function, and Genetics

Article Title: The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease

doi: 10.1002/(SICI)1097-0134(19990801)36:2%3C147::AID-PROT2%3E3.0.CO;2-3

Figure Lengend Snippet: Fig. 6. (A) shows the structure of the HIV-1 protease color- coded according to the magnitude of the stability constants per residue. In this figure blue denotes the most stable regions of the protein and the areas colored red the least stable regions. The stability constants were calculated with the COREX algo- rithm.31,34,35 (B) shows the calculated effects of acetyl pepstatin binding on the magnitude of the stability constants per residue. Blue denotes the residues most affected and red the residues least affected by binding. The figure was made using the program GRASP.39

Article Snippet: HIV-1 protease (at 0.3 mg/mL) and different concentrations of acetyl pepstatin (Peptides International, Louisville, KY) was examined for thermal denaturation at a scanning rate of 1°C/min, from 25°C to 85°C.

Techniques: Residue, Binding Assay