human tfeb gfp  (Addgene inc)

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 1. Peptides derived from transcription factor EB bind to CN in mouse brain lysates. (A) The TFEB-YLENP peptide binds CNA in mouse brain lysates in pull-down assays. Bound CNA was visualized by western blot with monoclonal anti-CNA antibody, shown in the upper blot; the input is shown in the middle panel. The bottom blot confirms that equal amounts of GST fusion proteins were used in the reactions. (B) Alterations of the TFEB-YLENP peptide decrease the interaction between peptides and CNA in mouse brain lysates assayed by GST pull-down. (C) The TFEB-YLENP derivatives TFEB-YLENPN78V and TFEB-YLENPE77A, N78V interact more strongly with CNA. (D) Densitometric quantification of CNA bound by TFEB-YLENPE77A and TFEB-YLENPE77A, N78V and histograms showing the relative intensity units of bound CN. Data were presented as mean ± SEM (n ¼ 3), *p < 0.05; **p < 0.01 compared with the TFEB-YLENP group.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Derivative Assay, Western Blot

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 2. Peptides derived from transcription factor EB bind to purified recombinant CN. (A) The affinity of FAM-labeled TFEB-YLENP peptide for CN. (B) The affinity of FAM- labeled TFEB-YLAVP peptide for CN. CN was titrated against fixed concentrations of labeled TFEB-YLENP (40 nM) and TFEB-YLAVP (20 nM). The top panel shows the isotherm derived from the raw data, fitted to a sigmoidal dose-response curve. The bottom panel displays the raw data for thermophoresis recorded at 20 C using 20% LED power and 80% MST power. (C) Comparison of Kd values between TFEB-YLENP/CN and TFEB-YLAVP/CN from three individual experiments. The individual experimental values were showed. (D) The interaction between CN and 300 nM FAM-labeled TFEB-YLAVP measured by fluorescence polarization. (E) Complex formation between CN and 300 nM FAM-labeled TFEB-YLAVP in the presence of unlabeled NFATc1-YLAVP peptide by fluorescence polarization.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Derivative Assay, Recombinant, Labeling, Comparison

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 3. A similar binding pattern for both TFEB-YLENP and LxVP-type peptides to CN. (A and B) The distance (upper) between C-alpha atoms of the peptide and CN, and their interaction energy (lower) during the simulations. (C) The YLENP (shiny) and YLAVP (brushed) peptides bind to CN in a similar manner, as shown in the last snapshots of the simulation. Both CNA (ice blue) and CNB (orange) are shown as transparent surfaces and cartoons, whereas the LxxP peptides are drawn as sticks. (D) Binding between CN and the TFEB-YLENP peptide is weaker than that between CN and the TFEB-YLAVP peptide, as shown by the average interaction energies based on the complete trajectories.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Binding Assay

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 4. CsA bound to cyclophilin competes with activated CN for the mutant TFEB. (A) Competition for binding of CN to TFEB-YLAVP by CsA alone and by the CsA-CyP complex (20 mM and 200 nM, respectively). (B) Binding of GST-CNA and its deletion mutants to GFP-TFEB in HeLa cells. (C) TFEB bound by CNA and its mutants was measured densito- metrically, and the histograms show the relative intensity units of bound TFEB. TFEB bound by CNA is set at 100%. Data were presented as mean ± SEM (n ¼ 3), *p < 0.05 compared with CNA group. (D) Quercetin (50 mM and 100 mM) competes with binding of GFP-TFEB to GST-CNA. (E) The bar graph depicts the ratios of bound TFEB and GST-CN in the presence of quercetin as percentages of the control binding (100%). Data were presented as mean ± SEM (n ¼ 3), **p < 0.01; ***p < 0.001 compared with the control group.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Mutagenesis, Binding Assay, Control

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 5. Expression of NFATc1-YLAVP peptide blocks TFEB activation in starved HeLa cells. (A) Starvation induces nuclear translocation of GFP-TFEB. (B) NFATc1-YLAVP peptide inhibits TFEB nuclear translocation. The graph shows the percentages of TFEB translocation in starved and peptide-treated cells. Data were presented as mean ± SEM (n ¼ 3), *p < 0.05; **p < 0.01 compared with the starved group. (C) Transfection of NFATc1-YLAVP cDNA induces TFEB phosphorylation in starved HeLa cells. Empty vector cDNA and NFATc1- YLAVP cDNA were transfected into HeLa cells, and the cells were starved. The blot shows the expression of GFP-TFEB in total lysates and indicates the positions of phosphorylated and dephosphorylated proteins.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Expressing, Activation Assay, Translocation Assay, Transfection, Phospho-proteomics, Plasmid Preparation