Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Primer sequences of TSPAN12.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Amplification

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Chromatograms and pedigrees of three families with familial exudative vitreoretinopathy. Three novel mutations were identified in TSPAN12 . A : In Family A, the affected mother and son had the c.566G>A (p.C189Y) mutation. C : In Family B, the patient and her affected mother had the c.177delC (p.Y59fsX67) mutation. E : In Family C, the affected mother and son had the c.C254T (p.T85M) mutation. The columns from left to right display the pedigree and the sequence chromatograms of these patients ( A , C , E ) and the normal controls ( B , D , F ). Arrows indicate the positions of the altered nucleotides.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Mutagenesis, Sequencing

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Protein sequence alignment of human TSPAN12 with its orthologs. The conserved amino acid residues are shaded. The orthologs are from the following species: Homo sapiens ( NP_036470 ), Pan troglodytes ( XP_001142754 ), Musmusculus ( NP_766595 ), Rattus norvegicus ( NP_001015026 ), Bos taurus ( NP_001039977 ), Equus caballus ( XP_001502093 ), Canis lupus familiaris ( XP_855095 ), Monodelphis domestica ( XP_001364876 ), Gallus gallus ( NP_001007850 ), Taeniopygia guttata ( XP_002192381 ), Ornithorhynchus anatinus ( XP_001516347 ), and Danio rerio (NP_957446). A : The residue of the missense mutation p.C189Y is highly conserved. B : The residue of the missense mutation p.T85M is also highly conserved.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Sequencing, Residue, Mutagenesis

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Luciferase assays with the SuperTopFlash cell line transfected with the indicated plasmids. SuperTopFlash (STF) cells/well were transfected with 800 ng DNA (200 ng of Norrin, 200 ng of FZD4, 200 ng of LRP5, 100 ng of pSV-β-galactosidase control vector, and 100 ng of TSPAN12 plasmid [wild-type or mutation]) and 1.5 µl Lipofectamine 2000 transfection reagent. Forty-eight hours after transfection, the cells were harvested and washed twice with PBS. Luciferase activities were measured with a dual-luciferase assay kit. Reporter activity was normalized to the coexpressed β-galactosidase activity in each well. Each test was performed in triplicate. The reporter assay was repeated three times, and a representative result was obtained.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Luciferase, Transfection, Control, Plasmid Preparation, Mutagenesis, Activity Assay, Reporter Assay

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Western blot analysis by SDS-PAGE of the TSPAN12 mutants. Total protein (10 μg) isolated from cell lysates from luciferase assays was mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer and subjected to SDS-PAGE and western blot analysis using anti-Flag antibody to detect TSPAN12 and FZD4 expression. Beta-actin was used as the loading control. The expression level of TSPAN12 C189Y was compatible with that of the wild-type. However, the T85M and Y59fs mutant proteins were not stable.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Western Blot, SDS Page, Isolation, Luciferase, Polyacrylamide Gel Electrophoresis, Expressing, Control, Mutagenesis

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Immunofluorescence staining of the TSPAN12 C189Y mutant. Cos 7 cells were transfected either with human wild-type or mutant TSPAN12 cloned into the pCMV6-entry vector, or empty vector. Cells were washed with PBS after 48 h and fixed with 4% PFA for 15 min. Mouse monoclonal anti-Flag antibody and Alexa Fluor 594 goat anti-mouse immunoglobulin (IgG) secondary antibody were used to detect TSPAN12 expression with the standard immunostaining method. Red channel, TSPAN12; blue channel, 4',6-diamidino-2-phenylindole (DAPI) for nuclei staining.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Immunofluorescence, Staining, Mutagenesis, Transfection, Clone Assay, Plasmid Preparation, Expressing, Immunostaining

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Diagram showing 9 known TSPAN12 mutations and three novel mutations identified in this study. The novel mutations are red.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 1. Immunohistochemical staining with Abs against CD40 (A), CD40L (B), IFN-g (C), and control Ig (D and E) in muscle tissue of PM/ DM. A, mAb against CD40 labeled the sarcolemma of muscle fibers (as- terisk), as well as endomysial MNCs (thick arrow) and endothelial cells of the capillary wall (thin arrow) obtained from a patient with PM (patient 1). CD40-positive fibers tended to be smaller in diameter than CD40-negative fibers. Similar results were observed in patients 2–9. B, Infiltrating MNCs surrounding muscle cells expressed CD40L (patient 1). Similar results were observed in patients 2–10. C, MNCs in the endomysium as well as the perivascular area expressed IFN-g in a patient with DM (patient 6). Similar results were observed in patients 1–4 and 7–9. D, The staining of control mouse IgG1. E, The staining of control rabbit IgG. Original magnification, 3400.

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Immunohistochemical staining, Staining, Control, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 3. A, CD40 mRNA expression in SkMC 2859. Total RNA was prepared from un- stimulated and IFN-g-stimulated SkMC 2859. cDNAs of CD40 and actin were amplified by RT-PCR. Product sizes for CD40 and b-actin genes are 430 and 218 bp, respectively. Lane 1, Unstimulated SkMC 2859; lane 2, IFN-g-stim- ulated SkMC 2859. B, Induction of CD40 ex- pression on SkMC 2859 by IFN-g and/or TNF-a. Upper panel, SkMC 2859 were stimu- lated with various concentrations of IFN-g for 48 h, stained with anti-CD40 mAb, and analyzed by flow cytometry (solid histogram). Fluores- cence levels for isotype-matched negative con- trol mAb are indicated by the dotted histogram. The x-axis represents the fluorescence intensity, and the y-axis shows the cell number. IFN-g in- duced CD40 expression on SkMC 2859 in a dose-dependent manner. Lower panel, Surface CD40 expression on resting or cytokine-stimu- lated SkMC 2859. SkMC 2859 were stimulated or unstimulated with TNF-a alone, IFN-g alone, or a combination of these cytokines for 48 h; stained with anti-CD40 Ab; and analyzed by flow cytometry (solid histogram). The back- ground fluorescence levels are shown by the dot- ted histogram. IFN-g and TNF-a showed a syn- ergistic effect on CD40 induction. p, Percentage of positive cells.

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 4. A, Effect of rhCD40L on cytokine production of SkMC 2859. SkMC 2859 were stimulated or unstimulated with 100 U/ml of IFN-g for 48 h to induce CD40 expression, and then cultured in medium containing various concentrations of rhCD40L for 48 h. At the end of culture, the culture supernatants and cell extracts were collected, and the concentrations of cytokines were determined by ELISA. The results are expressed as the mean 6 SE (n 5 3). f, without IFN-g, intracellular cytokines; M, without IFN-g, secreted cytokines; p, stimulated with IFN-g, intracellular cytokines; u, stimulated with IFN-g, secreted cytokines. p, p , 0.05; pp, p , 0.01. ND, not detectable. B, mRNA expression of cytokine in SkMC 2859. Total RNA was prepared from rhCD40L-stimulated SkMC 2859 that had been treated with 100 U/ml of IFN-g, and RT-PCR was performed as described in Materials and Methods. Amplified products for IL-6, IL-8, IL-15, MCP-1, and b-actin are shown. Product sizes of the genes for IL-6, IL-8, IL-15, and MCP-1 are 627, 292, 736, and 263 bp, respectively.

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 5. Expression of IL-6, IL-15, and MCP-1 in muscle tissue of PM/DM and normal controls. Some muscle cells were stained with anti- IL-6 mAb (A) or anti-MCP-1 mAb (C) in patient 2 (PM). These mAbs stained the cytoplasm of muscle cells. These IL-6- or MCP-1-positive mus- cle cells were smaller in diameter than muscle cells that were negative for stainings. Infiltrating MNCs also expressed MCP-1. Similar results were observed in patients 1, 6, and 7 (data not shown), but not in normal controls (F and H). The cytoplasm of muscle cells was strongly stained with anti- IL-15 mAb in patient 2 (B), although that of normal controls was margin- ally stained (G). Similar results were observed in patients 1, 6, and 7. D and E, Control staining of mouse IgG1 (D) and goat IgG (E) in patient 2. I and J, Control staining of mouse IgG1 (I) and goat IgG (J) in normal control. Original magnification, 3400

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Expressing, Staining, Control

Journal:

Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

doi: 10.1096/fj.09-133462

Figure Lengend Snippet: Membrane protein complexes with TSPAN12 and CD81

Article Snippet: cDNA for human TSPAN12 was amplified using reverse transcriptase (Promega, Madison, WI, USA) and Pfu polymerase (Stratagene, La Jolla, CA, USA).

Techniques:

Journal:

Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

doi: 10.1096/fj.09-133462

Figure Lengend Snippet: Confirmation of ADAM10-TSPAN12 association. A, B) Indicated FLAG-tagged proteins were immunoprecipitated from HT1080 (A) and MDA231 cells (B). ADAM10 (top panel) and FLAG-tagged proteins (bottom panel) were detected by immunoblotting. Pro-ADAM10, 95 kDa; mature ADAM10, ∼68 kDa. C) Endogenously expressed ADAM10 (MAB1427) and ADAM17 were immunoprecipitated from HT1080 cells, after cells were lysed in 0.5% Brij 97. Resulting proteins were detected using anti-ADAM10 polyclonal antibody. Presence of ADAM17 was confirmed using anti-ADAM17 polyclonal antibody (not shown). D) HT1080 cells stably expressing the indicated FLAG-tagged proteins were lysed in 0.5% Brij 97. Anti-FLAG immunoblotting was used to detect proteins in lysates (lanes 1–3) and in complexes after ADAM10 was immunoprecipitated using mAb MAB1427 (lanes 4–6). CD81 appears as dimer and monomer, as described previously (32). Ig(hc), immunoglobulin heavy chain; Ig(lc), immunoglobulin light chain.

Article Snippet: cDNA for human TSPAN12 was amplified using reverse transcriptase (Promega, Madison, WI, USA) and Pfu polymerase (Stratagene, La Jolla, CA, USA).

Techniques: Immunoprecipitation, Western Blot, Stable Transfection, Expressing

Journal:

Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

doi: 10.1096/fj.09-133462

Figure Lengend Snippet: TSPAN12 mutations variably disrupt ADAM10 association. A) HT1080 cells stably expressing FLAG-tagged proteins were lysed (0.5% Brij 97), and then ADAM10 (top panel) and FLAG-tagged proteins (bottom panel) were immunoprecipitated. Proteins were then detected by anti-FLAG immunoblotting (both panels). B) ADAM10 association is summarized for TSPAN12 mutants. SEL, small extracellular loop; LEL, large extracellular loop. Additional mutant information is in Supplemental Table S2.

Article Snippet: cDNA for human TSPAN12 was amplified using reverse transcriptase (Promega, Madison, WI, USA) and Pfu polymerase (Stratagene, La Jolla, CA, USA).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

Journal:

Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

doi: 10.1096/fj.09-133462

Figure Lengend Snippet: TSPAN12 expression influences ADAM10-dependent proteolysis of APP. A) Indicated FLAG-tagged proteins were expressed stably in MCF7 and SH-SY5Y cells; shed APP was detected by immunoblotting. B) Endogenous ADAM10 (siRNA = ADAM10–3), TSPAN12 (siRNA-1), CD81 (21) and CD9 (25) were knocked down, and shed APP was detected. Numbers below panels represent protein density scanning results.

Article Snippet: cDNA for human TSPAN12 was amplified using reverse transcriptase (Promega, Madison, WI, USA) and Pfu polymerase (Stratagene, La Jolla, CA, USA).

Techniques: Expressing, Stable Transfection, Western Blot

Journal:

Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

doi: 10.1096/fj.09-133462

Figure Lengend Snippet: TSPAN12 expression influences ADAM10 maturation. A) MCF7 cells expressing the indicated FLAG-tagged proteins were lysed (0.5% Brij 97), and ADAM10 and tubulin were detected by immunoblotting. The ∼85-kDa protein in the TSPAN12 lane likely represents an atypical form of ADAM10 that has been intermediately processed. B) MCF7 cells were treated with siRNAs (as in Fig. 3B), and then lysed and immunoblotted as in A. Numbers below panels represent ADAM10 active/prodomain ratios.

Article Snippet: cDNA for human TSPAN12 was amplified using reverse transcriptase (Promega, Madison, WI, USA) and Pfu polymerase (Stratagene, La Jolla, CA, USA).

Techniques: Expressing, Western Blot

Journal:

Article Title: Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein

doi: 10.1096/fj.09-133462

Figure Lengend Snippet: TSPAN12(Pal−) mutant associates with wild-type TSPAN12 and perturbs its subcellular distribution. A) TSPAN12(Pal−)-GFP (lanes 2–5, 7–10) or CD81(Pal−)-GFP (lanes 6, 11) were expressed in MCF7 cells, together with vector-FLAG (lanes 5, 10) or other FLAG-tagged proteins (lanes 2–4, 6, 8, 9, 11). Lanes 1 and 7 are from untransfected MCF7 (no GFP or FLAG proteins). After lysis (1% Triton X-100) MCF7 cells were treated with (lanes 7–11) or without (lanes 1–6) covalent cross-linker DSP. After anti-FLAG immunoprecipitations, reduction of the dithiol cross-link, and SDS-PAGE, proteins were detected by GFP immunoblotting. Diffuse proteins of 50–60 kDa in lanes 5, 9, and 10 likely represent background proteins, immunoprecipitated by anti-FLAG antibody (even when no FLAG proteins are present, as in lanes 5, 10), which also weakly cross-react with anti-GFP antibody. B) Either TSPAN12-GFP (a–c) or TSPAN12(Pal−)-GFP (d) was expressed in MCF7 cells, together with indicated FLAG-tagged proteins. FLAG- and GFP-tagged tetraspanins were expressed at comparable levels, as seen by immunoblotting (not shown).

Article Snippet: cDNA for human TSPAN12 was amplified using reverse transcriptase (Promega, Madison, WI, USA) and Pfu polymerase (Stratagene, La Jolla, CA, USA).

Techniques: Mutagenesis, Plasmid Preparation, Lysis, SDS Page, Western Blot, Immunoprecipitation