Journal: bioRxiv

Article Title: Inter-subunit Crosstalk via PDZ Synergistically Governs Allosteric Activation of Proapoptotic HtrA2

doi: 10.1101/2021.10.04.462974

Figure Lengend Snippet: Representative images from (A) Substrate-induced activation (B) Temperature-induced activation. (C) The fluorescence intensities of the control samples in (A) were quantified using the Image Lab TM software (version 6.0.0 build 25). The values obtained for each subunit from multiple independent experiments were plotted using Graphpad Prism software with their respective SEM. (D) The fluorescence intensities of the control samples in (B) were quantified and represented in a similar way as in (C).

Article Snippet: For better quantitative analysis, the fluorescence intensities of each subunit band were measured using Image Lab TM -version 6.0.0 build 25 (2017, Bio-Rad laboratories, Inc.) and the fold change between the intensities of the subunits in the test samples with respect to their corresponding controls were then plotted for graphical representation.

Techniques: Activation Assay, Fluorescence, Software

Journal: bioRxiv

Article Title: Inter-subunit Crosstalk via PDZ Synergistically Governs Allosteric Activation of Proapoptotic HtrA2

doi: 10.1101/2021.10.04.462974

Figure Lengend Snippet: (A) Active-site modification assay performed in presence of substrate. Each variant (2 µM) was reacted with TAMRA-FP (20 µM) in absence and presence of β-casein at 37 °C for 30 min. The reaction mixtures were run on SDS-PAGE, and fluorescence scan of the complete gel was taken using ChemiDoc™ MP Imaging System. The fluorescence intensities for the W and Δ subunits in each variant were quantified using the Image Lab TM software (version 6.0.0 build 25). The values obtained for each subunit from multiple independent experiments were averaged and the fold changes between the tests with respect to their corresponding controls were plotted using Graphpad Prism software. (B) Active-site modification assay performed at increased temperature. Each variant (2 µM) was reacted with TAMRA-FP (20 µM) and incubated either at 37 °C (Control) or at 50 °C (Test) for 30 min. The fluorescence imaging analysis and quantification were done similarly as in (A). (C) Quantitative assessment of proteolytic activity of HtrA2 variants with active-site mutation. Initial velocities V 0 of HtrA2 and its variants were calculated using FITC labeled–β casein as the substrate. The solid lines are the nonlinear least squares fit of the data to the Hill form of the Michaelis–Menten equation: Velocity = V max /[1 + (K 0.5 /[substrate]) n ] using Graphpad Prism software. (D) Model representing the trans -mediated allosteric communication between the adjacent subunits of HtrA2 trimeric ensemble.

Article Snippet: For better quantitative analysis, the fluorescence intensities of each subunit band were measured using Image Lab TM -version 6.0.0 build 25 (2017, Bio-Rad laboratories, Inc.) and the fold change between the intensities of the subunits in the test samples with respect to their corresponding controls were then plotted for graphical representation.

Techniques: Modification, Variant Assay, SDS Page, Fluorescence, Imaging, Software, Incubation, Activity Assay, Mutagenesis, Labeling