Journal: The Journal of Investigative Dermatology

Article Title: Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

doi: 10.1016/j.jid.2015.11.027

Figure Lengend Snippet: Clinical phenotype and GNA11 and GNAQ genotypes with percentage mosaicism (where available) of different samples from eight patients with PPV (patients 1–8) and three patients with extensive dermal melanocytosis with no vascular phenotype (patients 9–11), showing postzygotic mosaicism

Article Snippet: The human GNA11 cDNA clone was purchased from Origene (purchased from NM_002067.1 precloned into an untagged pCMV6-XL4 vector, catalog number SC303115).

Techniques: Biomarker Discovery, Mutagenesis

Journal: The Journal of Investigative Dermatology

Article Title: Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

doi: 10.1016/j.jid.2015.11.027

Figure Lengend Snippet: Sequencing results demonstrating mosaic GNA11 and GNAQ mutations. ( a ) Sanger sequencing of skin biopsy showing a very low peak in GNA11 at position c.547C>T (p.Arg183Cys) (asterisk). ( b ) Sanger sequencing of the same skin biopsy deoxyribonucleic acid after restriction enzyme digest of the normal allele, and hemi-nested amplification, revealing the mutation (asterisk). ( c ) Targeted next-generation sequencing showing low allele percentage mutations in skin but undetectable in blood, GNA11 c.547C>T (p.Arg183Cys) in 5% of reads. ( d ) GNAQ c.548G>A (p.Arg183Gln) in 6% of reads from skin but undetectable in blood.

Article Snippet: The human GNA11 cDNA clone was purchased from Origene (purchased from NM_002067.1 precloned into an untagged pCMV6-XL4 vector, catalog number SC303115).

Techniques: Sequencing, Amplification, Mutagenesis, Next-Generation Sequencing

Journal: The Journal of Investigative Dermatology

Article Title: Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

doi: 10.1016/j.jid.2015.11.027

Figure Lengend Snippet: Mutant GNA11 leads to activation of downstream signaling pathways. ( a ) Western blot for Flag, total GNA11, and the phosphorylated and total p38, JNK, ERK and AKT in total protein lysates from HEK293T cells transfected with either vector, wild-type GNA11 (WT), or one of two mutants R183C or Q209L mutant GNA11 . ( b ) Ratio of phosphorylated to total p38, JNK, ERK, and AKT normalized by actin loading control. Bars indicate 1 standard deviation. ∗ P < 0.05, unpaired t-test.

Article Snippet: The human GNA11 cDNA clone was purchased from Origene (purchased from NM_002067.1 precloned into an untagged pCMV6-XL4 vector, catalog number SC303115).

Techniques: Mutagenesis, Activation Assay, Protein-Protein interactions, Western Blot, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: The Journal of Investigative Dermatology

Article Title: Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

doi: 10.1016/j.jid.2015.11.027

Figure Lengend Snippet: Mosaic expression of GNA11 promotes ectopic pigmentary lesions in zebrafish. ( a ) Images of adult zebrafish mosaic for GNA11, GNA11 R183C , or GNA11 Q209L expression. Large, ectopic pigmentary lesions are indicated next to white arrows. Dashed box indicates zoomed areas that show detail of pigmentary lesions. ( b ) Numbers of pigmentary lesions per fish expressing GNA11, GNA11 R183C , or GNA11 Q209L . Dark circles indicate ectopic pigmentary lesions. White circles indicate fish without pigmentary lesions. ( c ) Histology hematoxylin and eosin staining of a wild-type and GNA11 R183C zebrafish skin at ×100 and ×400 magnification. Melanocytes are clearly visible in the dermis by the black melanin (blue arrows), frequently also in the epidermis (not shown) and in a few cases within underlying muscle (yellow arrow).

Article Snippet: The human GNA11 cDNA clone was purchased from Origene (purchased from NM_002067.1 precloned into an untagged pCMV6-XL4 vector, catalog number SC303115).

Techniques: Expressing, Staining

Journal: Immunity

Article Title: Immunization with components of the viral fusion apparatus elicits antibodies that neutralize Epstein-Barr virus in B cells and epithelial cells

doi: 10.1016/j.immuni.2019.03.010

Figure Lengend Snippet: Construction and characterization of gH/gL-ferritin and gH/gL/gp42-ferritin nanoparticles. (A) Schematic representation of full-length gH, soluble gH, gH-ferritin, full-length gp42, soluble gp42, and full-length gL. gH-ferritin fusion protein was generated by fusion of gH ectodomain (domains I, II, III, and IV) to the N-terminus of the ferritin. SS is the native signal sequence of the glycoprotein, SS* is the human CD5 signal sequence, TM is the transmembrane domain and CT is the cytoplasmic tail. Amino acid (aa) position of gH, gL, and gp42 is indicated. (B) Chromatograph from size-exclusion chromatography of gH/gL-ferritin and gH/gL/gp42-ferritin nanoparticles purified from the supernatants of mammalian cells transfected with gH-ferritin and gL plasmids or gH-ferritin, gL, and gp42 plasmids. Particles were affinity purified using snowdrop lectin prior to size exclusion chromatography. Transient transfection of gH-ferritin/gL and gH-ferritin/gL/gp42 plasmids, resulted in comparable amount of nanoparticle proteins (~ 2mg/L). (C) Characterization of nanoparticles by immunoprecipitation and SDS-PAGE. Bands corresponding to gH-ferritin, gp42, and gL are indicated. Purified nanoparticles were immunoprecipitated with anti-gH/gL mAb (E1D1), anti-gp42 mAb (F-2–1), anti-gp350 mAb (72A1), or isotype control antibody. HC and LC denote antibody heavy and light chains, respectively. Faint bands running slightly slower than gH-ferritin are nonspecific background bands. (D) Sandwich ELISA using mAb E1D1 or F-2–1 to capture purified gH/gL/gp42-ferritin nanoparticles and detection with mAb F-2–1 or E1D1, respectively. The dotted line represents the background. See also Figure S2 and S3.

Article Snippet: Proteins in the supernatant of cell culture were purified by GNA-Immobilized Lectin beads (EY Laboratories Inc.), and eluted proteins were purified by size exclusion chromatography using Superose 6 10/300 Gl and HiPrep 16/60 Sephacryl S-500 HR for soluble proteins and nanoparticles, respectively.

Techniques: Generated, Sequencing, Size-exclusion Chromatography, Purification, Transfection, Affinity Purification, Immunoprecipitation, SDS Page, Control, Sandwich ELISA

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins

doi: 10.3390/molecules22071104

Figure Lengend Snippet: Detection of ConA and GNA binding to glycoproteins in S, R and T cells passaged three times in the absence or presence of either tunicamycin (0.1 μmol·dm −3 ) or GalNAc-α- O -benzyl (0.1 mmol·dm −3 ). Panel ( a ): Representative sample of flow cytometry measurement of FITC-ConA binding to S (upper plot), R (middle plot) and T (lower plot) cells. Black histogram – unlabeled S, R and T cells, red histogram – FITC-ConA-labeled S cells, blue histogram FITC-ConA-labeled R cells, and green histogram – FITC-ConA-labeled T cells. Parameter Δ (represents the difference between medians of labeled and unlabeled cells) was ascertained using this measurement. Similar measurements were obtained for S, R and T cells untreated and treated with tunicamycin and GalNAc-α- O -benzyl and documented in panels ( b ) (for ConA) and ( c ) (for GNA). Binding of lectin to untreated S cells was arbitrarily selected as 100%. White column—untreated cells; gray—column cell treated with GalNAc-α- O -benzyl and black column—cell treated with tunicamycin. Significance: * and +—value significantly differs from the corresponding value for S cells at p < 0.02 and p < 0.05, respectively. The data represent the means ± S.E.M. of five independent measurements. Panels ( d ) (for ConA) and ( e ) (for GNA) represent Eastern blot identification of glycoproteins in crude membrane fractions isolated from S, R and T cells untreated C, or treated with inhibitor of O-glycosylation (GalNAc-α- O -benzyl, O) or N-glycosylation (tunicamycin, N). Data are representative of three independent measurements. Red arrows indicate the P-gp form glycosylated with saccharides that are GNA ligands.

Article Snippet: For glycoprotein detection by Eastern blots, ConA and GNA conjugated with biotin (EY Laboratories Inc., San Mateo, CA, USA), and avidin conjugated with horseradish peroxidase (Sigma-Aldrich, San Diego, CA, USA) was used.

Techniques: Binding Assay, Flow Cytometry, Labeling, Isolation