lc3b sepharose Search Results


lc3b sepharose  (GE Healthcare)
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GE Healthcare lc3b sepharose
ALS-FTLD-associated SQSTM1 mutations impact on the recognition of <t>LC3B</t> (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and <t>ubiquitin-Sepharose</t> (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.
Lc3b Sepharose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: In Vitro, Sequencing, Isolation, Incubation, Western Blot

ESI-MS indicates weaker binding of the LIR (L341V) to LC3B compared to WT LIR. (A) Native ESI-MS spectrum of an equimolar mixture of WT LIR and LIR (L341V) peptides (5 µM, residues 332 to 351). (B) LIR peptide mixture titrated with 5 µM LC3B. Top, full spectrum indicating free LIR (gray filled circle, mixture of WT LIR and LIR [L341V]), free LC3B and LC3B-LIR complexes of indicated (multiple) charge states. Below, zoomed-in spectra showing m/z values of the free and bound LIR complexes at the indicated charge states.

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: ESI-MS indicates weaker binding of the LIR (L341V) to LC3B compared to WT LIR. (A) Native ESI-MS spectrum of an equimolar mixture of WT LIR and LIR (L341V) peptides (5 µM, residues 332 to 351). (B) LIR peptide mixture titrated with 5 µM LC3B. Top, full spectrum indicating free LIR (gray filled circle, mixture of WT LIR and LIR [L341V]), free LC3B and LC3B-LIR complexes of indicated (multiple) charge states. Below, zoomed-in spectra showing m/z values of the free and bound LIR complexes at the indicated charge states.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay

Isothermal titration calorimetry (ITC) binding isotherms from titrating LC3B with LIR peptides at 25°C. Binding isotherms were fitted to a one-site binding model and K d values determined. Starting concentration of the LC3B was approximately 30 µM, and of LIR peptide (residues 332 to 351) stocks approximately 350 µM. Black diamond, LIR (L341V); gray circle WT LIR.

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) binding isotherms from titrating LC3B with LIR peptides at 25°C. Binding isotherms were fitted to a one-site binding model and K d values determined. Starting concentration of the LC3B was approximately 30 µM, and of LIR peptide (residues 332 to 351) stocks approximately 350 µM. Black diamond, LIR (L341V); gray circle WT LIR.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Isothermal Titration Calorimetry, Binding Assay, Concentration Assay

NMR titrations indicate selective perturbations of LC3B residues upon binding of LIR peptides. (A) 1 H- 15 N-HSQC spectrum of LC3B (0.25 mM dark gray) overlaid with the spectrum of the complex of LC3B with WT LIR (0.5 mM blue, residues 332 to 351) (ratio of 1:2) showing extensive chemical shift perturbations (CSPs) across the spectrum induced by ligand binding at 298 K; (B) Expansion of the region highlighted in (A) showing overlap between the spectrum of LC3B (dark gray), LC3B+WT LIR (blue) and LC3B + LIR (L341V) (green) illustrating a number of key residues within the LIR binding pocket that are perturbed to different extents by the WT LIR and LIR (L341V). Arrows identify the shifts of V33, F52 and V54 in the 2 complexes, while the majority of other residues show only small differences between the spectra of the 2 complexes (overlap of blue and green peaks), demonstrating selective effects on residues in direct contact with the LIRs.

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: NMR titrations indicate selective perturbations of LC3B residues upon binding of LIR peptides. (A) 1 H- 15 N-HSQC spectrum of LC3B (0.25 mM dark gray) overlaid with the spectrum of the complex of LC3B with WT LIR (0.5 mM blue, residues 332 to 351) (ratio of 1:2) showing extensive chemical shift perturbations (CSPs) across the spectrum induced by ligand binding at 298 K; (B) Expansion of the region highlighted in (A) showing overlap between the spectrum of LC3B (dark gray), LC3B+WT LIR (blue) and LC3B + LIR (L341V) (green) illustrating a number of key residues within the LIR binding pocket that are perturbed to different extents by the WT LIR and LIR (L341V). Arrows identify the shifts of V33, F52 and V54 in the 2 complexes, while the majority of other residues show only small differences between the spectra of the 2 complexes (overlap of blue and green peaks), demonstrating selective effects on residues in direct contact with the LIRs.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay, Ligand Binding Assay

Chemical shift mapping of the binding of the WT LIR and LIR (L341V) peptides (residues 332 to 351) to 15 N-LC3B. (A) Chemical shift mapping of the WT LIR. Weighted chemical shift pertubation (CSP) data showing the residues of 15 N-LC3B (0.25 mM) that are perturbed upon WT LIR binding at a final ratio of 1:2 (LC3B:LIR) at 298 K. All CSPs above 1.0 are indicated. (B) Difference in CSP effects between WT LIR and LIR (L341V) sequences from NMR titrations at a final ratio of 1:2 (LC3B:LIR) at 298 K. The indicated residues showed CSPs that are substantially different between the 2 complexes (greater in the WT LIR). (C) Representation of binding surface of LC3B with WT LIR, generated by highlighting residues determined from NMR chemical shift mapping experiments. LC3B residues in blue showed the greatest CSP values. Backbone representation of the LIR peptide also indicated (residues DDDWTHLS, 335 to 342 shown). (D) Residues showing substantially different CSPs from the NMR titrations of LC3B with WT LIR compared to LIR (L341V), highlighted on the LC3B structure. Note the close correlation with the binding cleft in proximity to L341 of the LIR peptide (site of L341V indicated).

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: Chemical shift mapping of the binding of the WT LIR and LIR (L341V) peptides (residues 332 to 351) to 15 N-LC3B. (A) Chemical shift mapping of the WT LIR. Weighted chemical shift pertubation (CSP) data showing the residues of 15 N-LC3B (0.25 mM) that are perturbed upon WT LIR binding at a final ratio of 1:2 (LC3B:LIR) at 298 K. All CSPs above 1.0 are indicated. (B) Difference in CSP effects between WT LIR and LIR (L341V) sequences from NMR titrations at a final ratio of 1:2 (LC3B:LIR) at 298 K. The indicated residues showed CSPs that are substantially different between the 2 complexes (greater in the WT LIR). (C) Representation of binding surface of LC3B with WT LIR, generated by highlighting residues determined from NMR chemical shift mapping experiments. LC3B residues in blue showed the greatest CSP values. Backbone representation of the LIR peptide also indicated (residues DDDWTHLS, 335 to 342 shown). (D) Residues showing substantially different CSPs from the NMR titrations of LC3B with WT LIR compared to LIR (L341V), highlighted on the LC3B structure. Note the close correlation with the binding cleft in proximity to L341 of the LIR peptide (site of L341V indicated).

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay, Generated