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