recombinant human importin beta  (Novus Biologicals)

Journal: Biochemistry and biophysics reports

Article Title: Acetylation of the nuclear localization signal in Ku70 diminishes the interaction with importin-α.

doi: 10.1016/j.bbrep.2022.101418

Figure Lengend Snippet: Fig. 1. Effect of substitution of lysine residues in the Ku70 NLS with ace tyl–lysine on binding to Impα. (A) The effect of amino acid substitutions in the Ku70 NLS on Impα binding. The binding activity of Ku70 NLS and its mutant peptides to Impα in the presence of Impβ was analyzed by probing immunoblots with antibodies against Impα. (B) Quantification of the pull-down assays pre sented in panel (A). Measurements of the blot band intensity were performed using ImageJ 1.52a. Each graph represents the relative intensity with Ku70 NLS WT defined as 100%. The error bars indicate the standard deviation from three independent experiments. *p < 0.05 significant differences from the case when the expected value was defined as 100.

Article Snippet: A binding assay was performed with 50 μl NLS-immobilized sepharose, 1 mg/ml bovine serum albumin, 0.1 μg recombinant human Impα2 (NBP1-78888; Novus Biologicals, Centennial, CO, USA), and 0.1 μg recombinant human Impβ (NBP1-78815; Novus Biologicals) in 0.5 ml transport buffer by incubation for 2 h at 4 ◦C with gentle rotation.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Western Blot, Standard Deviation

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Molecular interactions during nuclear transport are shown for NLS-containing cargo molecule (orange), importin α (light green), importin β (dark green), Ran (red), NTF2 (cyan), CAS (blue), immobile binging partners (cross hatched), microtubules (cylinders) and NPC (black). Freely diffusing molecules are shown in unshaded regions, molecules bound to medium, slow or immobile partners are shown in shaded regions in nucleus and cytoplasm.

Article Snippet: Nuclear transport proteins Recombinant human importin β, importin α, and NTF2 proteins were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: FCS analysis of nuclear transport proteins in vitro

Article Snippet: Nuclear transport proteins Recombinant human importin β, importin α, and NTF2 proteins were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Autocorrelation functions for APC and importin β labeled with Alexa Fluor 647 were measured in vitro and inside live cells and normalized. Autocorrelation data for both APC and importin β in vitro were fitted with a single decay time τ = 300 μs. Autocorrelation data for APC in vivo was fitted with a decay time τ = 1000 μs. Autocorrelation data for importin β in vivo required multiple decay times for an adequate fit.

Article Snippet: Nuclear transport proteins Recombinant human importin β, importin α, and NTF2 proteins were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Labeling, In Vitro, In Vivo

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Intrinsically fluorescent APC and Alexa Fluor 647 labeled importin β, importin α, NTF2, Ran, RanE70A, RanT24N and RanQ69L were microinjected into the cytoplasm of B104 cells. After 30–60 minutes the injected cells were imaged by confocal microscopy. Scale bar indicates 10 μm.

Article Snippet: Nuclear transport proteins Recombinant human importin β, importin α, and NTF2 proteins were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Labeling, Injection, Confocal Microscopy

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Fluorescently labeled importin β, importin α, NTF2, Ran, RanE70A, RanT24N and RanQ69L labeled with Alexa Fluor 647 were microinjected into cytoplasm of B104 cells. After injected protein achieved steady state nucleocytoplasmic distribution, FCS measurements were performed with confocal volume positioned inside nucleus. For each protein at least 30 cells were analyzed. Autocorrelation data from all cells were globally fitted to resolve protein populations with different mobilities. Immobile fraction for each protein was determined from photobleaching amplitudes. Concentrations for each population were calculated based on total concentration and nucleocytoplasmic distribution of endogenous protein. Concentrations of each component are shown on bar graphs and in the table. Fast, medium, slow and immobile populations are labeled with red, green, blue, and black colors respectively. For mutant Ran proteins, the fraction of each population is shown compared with wt Ran. The table shows autocorrelation decay times (τD), fractions, and concentrations for each population. Molecular species comprising each population and calculated diffusion coefficients or off rates are listed.

Article Snippet: Nuclear transport proteins Recombinant human importin β, importin α, and NTF2 proteins were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Labeling, Injection, Concentration Assay, Mutagenesis, Diffusion-based Assay

Journal:

Article Title: SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY

doi: 10.1016/j.jmb.2006.09.08

Figure Lengend Snippet: Importin β, importin α, NTF2, Ran wt, RanE70A, RanT24N and RanQ69L labeled with Alexa Fluor 647 were microinjected into the cytoplasm of B104 cells. Cells were incubated for 30–60 minutes after injection and analyzed by FCS with observation volume positioned over the nuclear envelope. For each protein at least 20 cells were analyzed. To resolve protein populations specific to NPC and nuclear envelope, contributions from adjacent nucleoplasm and cytoplasm were subtracted by a global fitting procedure. Number of molecules per NPC for each protein component was calculated based on number of pores encompassed by FCS observation volume. Distributions of each protein between populations with different mobilities are shown on bar graphs and in the table. Fast, medium, slow and immobile populations are labeled with red, green, blue, and black colors respectively. For mutant Ran proteins, the fraction of each component compared with wt Ran is shown. The table shows autocorrelation decay times (τD), concentrations, numbers of molecules per NPC, or fractions for each population. Molecular species comprising each population and calculated diffusion coefficients or off rates are listed.

Article Snippet: Nuclear transport proteins Recombinant human importin β, importin α, and NTF2 proteins were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Labeling, Incubation, Injection, Mutagenesis, Diffusion-based Assay