Journal: Nature chemical biology

Article Title: Diabetes reversal by inhibition of the low molecular weight tyrosine phosphatase

doi: 10.1038/nchembio.2344

Figure Lengend Snippet: (a) Surface representation of human LMPTP-A showing phosphate (P) non-covalently bound in the active-site. Residues are colored according to magnitude of shift in the HSQC 15 N- 1 H spectrum upon Compd. 18 titration (red>orange>green). Gray residues had negligible shifts or could not be assigned. (b) Crystal structure of bovine LMPTP W49Y/N50E bound to orthovanadate and Compd. 18 (cyan and blue sticks; Q=quinoline; Pip=piperidine; BN=benzonitrile; L=linker), with selected side-chains (yellow=carbon; red=oxygen; blue=nitrogen; pink=vanadium) and H-bonds/ionic interactions (dashed green/gray lines) shown. (c) Inhibition of phosphatase activity of LMPTP-A/mutants by Compd. 18 using 0.4 mM OMFP substrate. Mean±SD % activity is shown. Data is representative of 3 independent experiments. (d) Compd. 18 modeled into the crystal structure of phosphate-bound human LMPTP, based on an overlay with the bovine ternary complex crystal structure (RMSD=0.33 Å). Selected residues are colored by NMR shift as in (a) . Dashed red line depicts predicted clash between apical oxygen (“A”) of phosphate and Q. (e–f) Structural rationale for SAR data, with atoms at 66% of their true radii. (e) “Side” view of pocket, rotated ~90° about a horizontal axis. The molecular surface has been sliced through the active-site to reveal the tight fit of Q in the pocket. Atoms with a formal charge (±) are labeled. BN is highly polarized, as indicated (δ±); arrows labeled “S” indicate solvent exposure of ring substitutions. (f) “Top” view looking down at the active-site pocket filled by Q. Arrow above atom N1 locates the “saddle-point” at pocket exit.

Article Snippet: cDNAs encoding mouse and human LMPTP-A (protein reference sequences NP_067305.2 and NP_004291.1) and bovine LMPTP (protein reference sequence NP_776403.1) were codon-optimized for E. coli , synthesized, and cloned into the pGEX-4T vector using BamHI/EcoRI by Genscript.

Techniques: Titration, Inhibition, Activity Assay, Labeling, Solvent

Journal: Neuron

Article Title: Age-dependent effects of apoE reduction using antisense oligonucleotides in a model of β-amyloidosis

doi: 10.1016/j.neuron.2017.11.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level was used as a reference (Mm99999915_g1 Gapdh).

Techniques: Purification, Virus, Recombinant, Bicinchoninic Acid Protein Assay, Sequencing, Control, Software

Journal: PLOS ONE

Article Title: Visfatin (NAMPT) expression in human placenta cells in normal and pathological conditions and its hormonal regulation in trophoblast JEG-3 cells

doi: 10.1371/journal.pone.0310389

Figure Lengend Snippet: Gene (A) and protein expression (B) of visfatin (p < 0.05) as well as its immunolocalization in JEG-3, BeWo cell lines and commercially human term placenta slides (C) (scale bar is 20 μm). Immunolocalization: cytoplasm of cells (arrows), villous cells (arrowheads), blood spaces surrounding the villi (asterisks). Fig 2B shows data from multiple blot images. The relative gene expression of visfatin was examined by qRT-PCR then the obtained results were normalized using the geometric mean of reference gene expression (GAPDH, TBP, YWHAZ) due to the comparative cycle threshold method. The protein expression of visfatin was detected by Western blot, and protein lanes were densitometrically measured and shown as the ratio relative to ACTB expression. Statistical analysis was shown using ANOVA followed by Tukey’s HSD multiple range test (mean ± SEM, p < 0.05; n = 3). ACTB—β-actin; NC—negative control.

Article Snippet: The reaction was performed under the following cycle conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. Gene expression was normalized using the geometric mean of three reference genes: GAPDH (cat. no. Hs02786624; RefSeq NM_001256799.2, Thermo Fisher Scientific, USA), TBP (cat. no. Hs00920495_m1; RefSeq NM_001172085.1, Thermo Fisher Scientific, USA), and YWHAZ (cat. no. Hs01122445_g1; RefSeq NM_09111, Thermo Fisher Scientific, USA) using the 2 -ΔCt method [ ].

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot, Negative Control

Journal: PLOS ONE

Article Title: Visfatin (NAMPT) expression in human placenta cells in normal and pathological conditions and its hormonal regulation in trophoblast JEG-3 cells

doi: 10.1371/journal.pone.0310389

Figure Lengend Snippet: Gene (A) and protein expression (B) of visfatin (p < 0.05, mean ± SEM) as well as its immunolocalization in term placenta from normal and IUGR, PE, GDM pregnancies (C) (scale bar is 50 μm). Immunolocalization: capillary epithelium (long arrows), decidual cells (double arrowheads), syncytiotrophoblasts (double arrowheads). Fig 3B shows data from multiple blot images. The relative gene expression of visfatin was examined by qRT-PCR then the obtained results were normalized using the geometric mean of reference gene expression (GAPDH, TBP, YWHAZ) due to the comparative cycle threshold method. The protein expression of visfatin was detected by Western blot, and protein lanes were densitometrically measured and shown as the ratio relative to ACTB expression. Statistical analysis was shown using ANOVA followed by Tukey’s HSD multiple range test (mean ± SEM, p < 0.05; n = 5). NP—normal placenta; IUGR—intrauterine growth restriction; PE—preeclampsia; GDM—gestational diabetes mellitus; NC—negative control; ACTB—β-actin.

Article Snippet: The reaction was performed under the following cycle conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. Gene expression was normalized using the geometric mean of three reference genes: GAPDH (cat. no. Hs02786624; RefSeq NM_001256799.2, Thermo Fisher Scientific, USA), TBP (cat. no. Hs00920495_m1; RefSeq NM_001172085.1, Thermo Fisher Scientific, USA), and YWHAZ (cat. no. Hs01122445_g1; RefSeq NM_09111, Thermo Fisher Scientific, USA) using the 2 -ΔCt method [ ].

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot, Negative Control

Journal: STAR Protocols

Article Title: Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

doi: 10.1016/j.xpro.2022.101785

Figure Lengend Snippet:

Article Snippet: Download the appropriate sequences. a. Download the Reference Sequence for Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA (GenBank: NM_058243.3) from the NCBI’s website, using the GenBank data base ( BRD4-L RefSeq ). b. Download the sequence for LentiV_Blast (Addgene, cat# 111887) from Addgene’s website ( LentiV_Blast ).

Techniques: Virus, Recombinant, Cloning, Gel Extraction, Plasmid Preparation, Sequencing, Over Expression, Expressing, Software, Imaging