flag ha tagged bap1 variant plasmids  (Addgene inc)

Journal: Chembiochem

Article Title: Characterization of BRCA1‐Associated Protein‐1 (BAP1) Aggregation Properties Induced by Cancer‐Associated Mutations

doi: 10.1002/cbic.202500372

Figure Lengend Snippet: Aggregation kinetics and model analysis by ThT binding assay. A) Aggregation kinetics of WT, N78S, C91W, F81V, and G128R BAP1‐UCH variants. The samples were tested in triplicate, with concentrations ranging from 30 μM to 1 μM, which are shown as dark to light colors. B) Half‐time plot of aggregation kinetics. The x ‐axis represents sample concentration, and the y ‐axis represents aggregation half‐time. Triplicates used in calculating slopes to explain the relationship between sample concentration and aggregation half‐time are marked with black borders. C) The aggregation mechanism is based on a secondary nucleation‐dominated model. k n , primary nucleation rate constant, k + , elongation rate constant, k 2 , secondary nucleation rate constant; n c , reaction order of primary nucleation; and n 2 , reaction order of secondary nucleation.

Article Snippet: HeLa‐Kyoto cells (Research Resource Identified, RRID: CVCL_1922; kindly provided by Professor Eric Nigg, University of Basel, Switzerland) grown on glass coverslips were transfected with FLAG‐HA‐tagged BAP1 variant plasmids ( https://www.addgene.org/22539/ ) for 48 h, fixed in buffer containing 20 mM PIPES (pH 6.8), 0.2% Triton X‐100, 10 mM EGTA, 1 mM MgCl 2 , and 4% formaldehyde (PTEMF solution), and then washed with phosphate‐buffered saline containing 0.05% Tween‐20 (PBST).

Techniques: Binding Assay, Concentration Assay

Journal: Chembiochem

Article Title: Characterization of BRCA1‐Associated Protein‐1 (BAP1) Aggregation Properties Induced by Cancer‐Associated Mutations

doi: 10.1002/cbic.202500372

Figure Lengend Snippet: Oligomerization kinetics of BAP1‐UCH variants. The hydrodynamic radii (R h ) of BAP1‐UCH variants as a function of incubation time monitored by DLS.

Article Snippet: HeLa‐Kyoto cells (Research Resource Identified, RRID: CVCL_1922; kindly provided by Professor Eric Nigg, University of Basel, Switzerland) grown on glass coverslips were transfected with FLAG‐HA‐tagged BAP1 variant plasmids ( https://www.addgene.org/22539/ ) for 48 h, fixed in buffer containing 20 mM PIPES (pH 6.8), 0.2% Triton X‐100, 10 mM EGTA, 1 mM MgCl 2 , and 4% formaldehyde (PTEMF solution), and then washed with phosphate‐buffered saline containing 0.05% Tween‐20 (PBST).

Techniques: Incubation

Journal: Chembiochem

Article Title: Characterization of BRCA1‐Associated Protein‐1 (BAP1) Aggregation Properties Induced by Cancer‐Associated Mutations

doi: 10.1002/cbic.202500372

Figure Lengend Snippet: Correlation plots of experimental folding stabilities of BAP1‐UCH variants with respect to the theoretical prediction of folding stability and aggregation kinetics parameters. The enthalpies of unfolding of the first transition, ΔH , and the second transition, ΔH 2 , of BAP1‐UCH variants, previously derived from DSC measurements, are shown on the horizontal axes. The theoretical free energy differences between the mutant and WT, ΔΔG mut‐WT , predicted by FoldX, are shown on the X ‐axes of A,B). The identities of the individual mutants are indicated next to the data point. Asterisks highlight the main mutants discussed in this study. The primary nucleation rate in logarithmic scales is shown in filled circles in C,D), with their scales indicated on the left Y ‐axes. The reaction order of the primary nucleation is shown in open squares in (C) and (D), with their scales shown on the right Y ‐axes. The secondary nucleation rate in logarithmic scales is shown in filled circles in E,F), with their scales indicated on the left Y ‐axes. The reaction order of the secondary nucleation is shown in open squares in (E) and (F), with their scales shown on the right Y ‐axes. The error bars represent the standard deviation (SD) of five replicates for each parameter as reported in Table .

Article Snippet: HeLa‐Kyoto cells (Research Resource Identified, RRID: CVCL_1922; kindly provided by Professor Eric Nigg, University of Basel, Switzerland) grown on glass coverslips were transfected with FLAG‐HA‐tagged BAP1 variant plasmids ( https://www.addgene.org/22539/ ) for 48 h, fixed in buffer containing 20 mM PIPES (pH 6.8), 0.2% Triton X‐100, 10 mM EGTA, 1 mM MgCl 2 , and 4% formaldehyde (PTEMF solution), and then washed with phosphate‐buffered saline containing 0.05% Tween‐20 (PBST).

Techniques: Derivative Assay, Mutagenesis, Standard Deviation