Journal: Frontiers in Physiology

Article Title: Characterization of MaltOBP1, a Minus-C Odorant-Binding Protein, From the Japanese Pine Sawyer Beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae)

doi: 10.3389/fphys.2020.00212

Figure Lengend Snippet: Structural analysis of MaltOBP1. (A) The secondary structure of MaltOBP1. (B) The 3D structure of MaltOBP1. The six α-helices are labeled as α 1 –α 6 . DB-I, Disulfide bridge-I; DB-II, Disulfide bridge-II.

Article Snippet: Recombinant Protein Expression and Purification The recombinant MaltOBP1 gene (without signal peptide) was inserted into Escherichia coli expression vector pET32a (Novagen, Madison, WI, United States) with N-terminus 6 × His tag and subsequent TEV cleavage site using recombinant PCR.

Techniques: Labeling

Journal: Frontiers in Physiology

Article Title: Characterization of MaltOBP1, a Minus-C Odorant-Binding Protein, From the Japanese Pine Sawyer Beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae)

doi: 10.3389/fphys.2020.00212

Figure Lengend Snippet: Sex- and tissue-expression profile of MaltOBP1. (A) Electrophoretic analysis of soluble proteins from Monochamus alternatus under 15% native PAGE. M1: Protein molecular weight marker. (B) Recombinant protein analyzed by SDS-PAGE. M2: Protein molecular weight marker; Lane 1: Non-induced pET32a-MaltOBP1 in Escherichia coli ; Lane 2: Expressed protein pET32a-MaltOBP1-His after induction by IPTG; Lane 3: pET32a-MaltOBP1 protein purified through Ni-NTA column; (C) Western blot analysis of MaltOBP1 expression in total protein extracts of male and female adults of M. alternatus . AT, antennae; H, head; T, thorax; AD, abdomen; L, leg; W, wing; OBP1, Recombinant MaltOBP1.

Article Snippet: Recombinant Protein Expression and Purification The recombinant MaltOBP1 gene (without signal peptide) was inserted into Escherichia coli expression vector pET32a (Novagen, Madison, WI, United States) with N-terminus 6 × His tag and subsequent TEV cleavage site using recombinant PCR.

Techniques: Expressing, Clear Native PAGE, Molecular Weight, Marker, Recombinant, SDS Page, Purification, Western Blot

Journal: Frontiers in Physiology

Article Title: Characterization of MaltOBP1, a Minus-C Odorant-Binding Protein, From the Japanese Pine Sawyer Beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae)

doi: 10.3389/fphys.2020.00212

Figure Lengend Snippet: Binding affinity of MaltOBP1. (A) Binding curve and relative Scatchard plot of MaltOBP1 and 1-NPN. (B) Competitive binding curves of selected volatile host plant compounds to MaltOBP1.

Article Snippet: Recombinant Protein Expression and Purification The recombinant MaltOBP1 gene (without signal peptide) was inserted into Escherichia coli expression vector pET32a (Novagen, Madison, WI, United States) with N-terminus 6 × His tag and subsequent TEV cleavage site using recombinant PCR.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

doi: 10.1371/journal.pone.0071035

Figure Lengend Snippet: Phosphorylation of Y416 in the closed, repressed conformation is driven by core kinase domains. A. Confocal micrographs of c-Src expressed as fusions to Emerald in AD293 cells in either full length or truncated form lacking the first 63 residues (Δ1–63), which contains the unique domain and myristoylation sequences. Cells were co-transfected with mKate2-F, which is a fluorescent protein containing a farnesylation signal to localize it to membranes. Turquoise shows c-Src-Em, red shows mKate2-F. Scale bar, 10 µm. Images are representative of three independent experiments. B. Western Blots of AD293 cells transfected with the indicated constructs for 24 h with 10 µg total protein lysate. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. Key: Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs. Experiments were performed at least twice, which showed consistent results.

Article Snippet: The mKate2-F sequence was commercially synthesized (GeneArt) with human optimized codons and appended at the C-terminus with the sequence SGLRTKLNPPDESGPGCMSCKCVLS, which includes C-terminal 20 amino acid farnesylation signal sequence from the c-HA-Ras protein , .

Techniques: Transfection, Western Blot, Construct, Mutagenesis, Plasmid Preparation

Journal: Nature methods

Article Title: A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

doi: 10.1038/nmeth.2413

Figure Lengend Snippet: Fluorescence imaging of mNeonGreen (mNG) fusion vectors. C-terminal mNG fusion constructs (with respect to the fluorescent protein); for each fusion, the linker amino acid length is indicated after the name of the targeted organelle or fusion partner: ( a ) mNG-Annexin (A4)-C-12 (human; plasma membrane); ( b ) mNG-β-actin-C-18 (human; actin cytoskeleton); ( c ) mNG-β-Catenin-C-20 (mouse; tight junctions); ( d ) mNG-CAAX-C-5 (20-amino acid farnesylation signal from c-Ha-Ras; plasma membrane); (e) mNG-CAF1-C-10 (mouse chromatin assembly factor); ( f ) mNG-Caveolin1-C-10 (human); ( g ) mNG-Endosomes-C-14 (human RhoB GTPase); ( h ) mNG-Fascin-C-10 (human; actin bundling); ( i ) mNG-Fibrillarin-C-7 (human; nucleoli); ( j ) mNG-FilaminA-C-14 (human; cytoskeleton); ( k ) mNG-LAMP1-C-20 (rat; lysosomal membrane glycoprotein 1; lysosomes); ( l ) mNG-Clathrin-C-15 (human, light chain B); ( m ) mNG-Myotilin-C-14 (human; actin filaments); ( n ) mNG-PCNA-C-19 (human; replication foci); ( o ) mNG-Plastin-C-10 (human; actin binding); ( p ) mNG-Rab4a-C-7 (human; endosomes); ( q ) mNG-LC3B-C-7 (rat light chain; autophagosoms; ( r ) mNG-Talin-C-18 (mouse; focal adhesions); ( s ) mNG-Tubulin-C-35 (human; microtubules); ( t ) mNG-ZO1-C-14 (human; tight junctions). The cell line used for expression of C-terminal mNG constructs was Madin-Darby canine kidney (MDCK; ATCC, CCL-34) cells in panels ( c ) and ( t ). HeLa CCL2 (ATCC) cells were used in the remaining panels. Scale bars represent 10 μm.

Article Snippet: Thus, to prepare mNeonGreen C-terminal fusions (number of linker amino acids in parenthesis), the following digests were performed: annexin A4 (12), NheI and BspEI (Alen Piljic, EMBL, Heidelberg, Germany; NM_001153.3); β-actin (7), NheI and BglII (human β-actin cDNA source: Clontech; NM_001101.3); β-catenin (20), XhoI and BamHI (mouse β-catenin cDNA source: Origene, Rockville, MD; NM_001165902.1); 20 amino acid farnesylation signal from c-Ha-Ras (CAAX; 5), AgeI and BspEI (c-Ha-Ras cDNA source: Clontech; NM_001130442.1); CAF1 (10), AgeI and BspEI (mouse chromatin assembly factor cDNA source: Akash Gunjan, Florida State University; NM_013733.3); caveolin 1 (10), NheI and BglII (human caveolin 1 cDNA source: Origene; NM_001753); endosomes (14), NheI and BspEI (endosomes cDNA source: Clontech; NM_004040.2); fascin (10), BspEI and BamHI (human fascin cDNA source: Origene; NM_003088.2); fibrillarin (7), AgeI and BspEI (fibrillarin cDNA source: Evrogen, Moscow, Russia; NM_001436.3); filamin A (14), BspEI and HindIII (human filamin cDNA source: David Calderwood, Yale University; NM_001456.3); human lysosomal membrane glycoprotein 1 (20), BamHI and NotI (LAMP1; George Patterson, NIH, Bethesda MD, U.S.A.; NM_012857.1); human light chain clathrin (15), NheI and BglII (human clathrin light chain cDNA source: George Patterson, NIH; NM_001834.2); human myotilin, AgeI and BspEI (MYOT; Origene; NM_006790.1); PCNA (19), AgeI and BspEI (proliferating cell nuclear antigen cDNA source: David Gilbert, FSU; NM_002592.2); plastin (10), BspEI and XhoI (human plastin 1 (fimbrin) cDNA source: Origene; NM_002670.1); canine Rab4a, BglII and BamHI (Rab4a cDNA source: Viki Allen, U. Manchester, UK; NM_004578.2); LC3B (7), AgeI and BspEI (rat LC3B cDNA source: Jenny M. Tam, Harvard University; U05784.1); talin (22) AgeI and BspEI (mouse talin 1 cDNA source: Clare Waterman, NIH; NM_011602.5); α-tubulin (18), NheI and BglII (human α-tubulin cDNA source: Clontech; NM_006082).

Techniques: Fluorescence, Imaging, Construct, Binding Assay, Expressing